Isolated human enzyme proteins, nucleic acid molecules encoding human enzyme proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the enzyme peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the enzyme peptides, and methods of identifying modulators of the enzyme peptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of enzyme proteins that are related to the synthase enzyme subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides a novel alternative splice form of a squalene synthase enzyme and nucleic acid molecules encoding the novel splice form, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION

[0002] Many human enzymes serve as targets for the action of pharmaceutically active compounds. Several classes of human enzymes that serve as such targets include helicase, steroid esterase and sulfatase, convertase, synthase, dehydrogenase, monoxygenase, transferase, kinase, glutanase, decarboxylase, isomerase and reductase. It is therefore important in developing new pharmaceutical compounds to identify target enzyme proteins that can be put into high-throughput screening formats. The present invention advances the state of the art by providing novel human drug target enzymes related to the synthase subfamily.

[0003] Synthases

[0004] The novel human protein, and encoding gene, provided by the present invention is related to the family of synthases in general and squalene synthases (also known by such names as squalene synthetases and farnesyl-diphosphate farnesyltransferases) in particular. Furthermore, the protein of the present invention may be a novel isoform of the protein/gene provided in Genbank gi4758350. Specifically, the protein/cDNA of the present invention differs from the art-known protein of gi4758350 in that the fourth exon is spliced out of the protein/cDNA of the present invention (see the amino acid sequence alignments in FIG. 2).

[0005] Squalene synthase is important for catalyzing the first specific step in the biosynthesis of cholesterol, which is the conversion of trans-farnesyldiphosphate to squalene. Squalene synthase regulates this major control point in sterol, as well as isoprene, biosynthesis in eukaryotes (Robinson et al., Mol Cell Biol 1993 May;13(5):2706-17). Squalene synthase thus occupies a critical regulatory position in cholesterol synthesis (Schechter et al., Genomics March. 1, 1994;20(1):116-8). Furthermore, the squalene synthase gene has been associated with Rec syndrome (Patterson et al., Am. J. Hum. Genet. 57: A91 only, 1995).

[0006] Loss of promoter activity and response to sterols has been localized to a 69-bp region that is positioned 131 bp upstream from the transcription start site; this region contains a sterol regulatory element-1, which is found in other sterol regulated genes, and two possible NF1 binding sites (Guan et al., Biol. Chem. 270: 21958-21965, 1995).

[0007] For a further review of squalene synthases, see McKenzie et al., J Biol Chem Oct. 25, 1992;267(30):21368-74; Summers et al., Gene Dec. 22, 1993;136(1-2Che):185-92; Jiang et al., J Biol Chem Jun. 15, 1993;268(17):12818-24; and Soltis et al., Arch Biochem Biophys Feb. 1, 1995;316(2):713-23.

[0008] Due to their importance in cholesterol biosynthesis, novel human squalene synthase proteins/genes, such as provided by the present invention, are valuable as potential targets for the development of therapeutics to treat cholesterol-related diseases/disorders such as cardiovascular diseases. Furthermore, SNPs in squalene synthase genes, such as provided by the present invention, may serve as valuable markers for the diagnosis, prognosis, prevention, and/or treatment of cholesterol-related diseases/disorders such as cardiovascular diseases.

[0009] Using the information provided by the present invention, reagents such as probes/primers for detecting the SNPs or the expression of the protein/gene provided herein may be readily developed and, if desired, incorporated into kit formats such as nucleic acid arrays, primer extension reactions coupled with mass spec detection (for SNP detection), or TaqMan PCR assays (Applied Biosystems, Foster City, Calif.).

[0010] Enzyme proteins, particularly members of the synthase enzyme subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of enzyme proteins. The present invention advances the state of the art by providing previously unidentified human enzyme proteins, and the polynucleotides encoding them, that have homology to members of the synthase enzyme subfamily. These novel compositions are useful in the diagnosis, prevention and treatment of biological processes associated with human diseases.

SUMMARY OF THE INVENTION

[0011] The present invention is based in part on the identification of amino acid sequences of human enzyme peptides and proteins that are related to the synthase enzyme subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate enzyme activity in cells and tissues that express the enzyme. Experimental data as provided in FIG. 1 indicates expression in humans in placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, bladder, and liver.

DESCRIPTION OF THE FIGURE SHEETS

[0012]FIG. 1 provides the nucleotide sequence of a cDNA sequence that encodes the enzyme protein of the present invention. (SEQ ID NO:1) In addition, structure and functional information is provided, such as ATG start, stop and tissue distribution, where available, that allows one to readily determine specific uses of inventions based on this molecular sequence. Experimental data as provided in FIG. 1 indicates expression in humans in placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, bladder, and liver.

[0013]FIG. 2 provides the predicted amino acid sequence of the enzyme of the present invention. (SEQ ID NO:2) In addition structure and functional information such as protein family, function, and modification sites is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence.

[0014]FIG. 3 provides genomic sequences that span the gene encoding the enzyme protein of the present invention. (SEQ ID NO:3) In addition structure and functional information, such as intron/exon structure, promoter location, etc., is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence. As illustrated in FIG. 3, SNPs were identified at 55 different nucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION

[0015] General Description

[0016] The present invention is based on the sequencing of the human genome. During the sequencing and assembly of the human genome, analysis of the sequence information revealed previously unidentified fragments of the human genome that encode peptides that share structural and/or sequence homology to protein/peptide/domains identified and characterized within the art as being a enzyme protein or part of a enzyme protein and are related to the synthase enzyme subfamily. Utilizing these sequences, additional genomic sequences were assembled and transcript and/or cDNA sequences were isolated and characterized. Based on this analysis, the present invention provides amino acid sequences of a novel human synthase enzyme alternative splice form, nucleic acid sequences in the form of transcript sequences, cDNA sequences and/or genomic sequences that encode this novel enzyme splice form, nucleic acid variation (allelic information), tissue distribution of expression, and information about the closest art known protein/peptide/domain that has structural or sequence homology to the enzyme of the present invention.

[0017] In addition to being previously unknown, the peptides that are provided in the present invention are selected based on their ability to be used for the development of commercially important products and services. Specifically, the present peptides are selected based on homology and/or structural relatedness to known enzyme proteins of the synthase enzyme subfamily and the expression pattern observed. Experimental data as provided in FIG. 1 indicates expression in humans in placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, bladder, and liver. The art has clearly established the commercial importance of members of this family of proteins and proteins that have expression patterns similar to that of the present gene. Some of the more specific features of the peptides of the present invention, and the uses thereof, are described herein, particularly in the Background of the Invention and in the annotation provided in the Figures, and/or are known within the art for each of the known synthase family or subfamily of enzyme proteins.

[0018] Specific Embodiments

[0019] Peptide Molecules

[0020] The present invention provides nucleic acid sequences that encode protein molecules that have been identified as being members of the enzyme family of proteins and are related to the synthase enzyme subfamily (protein sequences are provided in FIG. 2, transcript/cDNA sequences are provided in FIG. 1 and genomic sequences are provided in FIG. 3). The peptide sequences provided in FIG. 2, as well as the obvious variants described herein, particularly allelic variants as identified herein and using the information in FIG. 3, will be referred herein as the enzyme peptides of the present invention, enzyme peptides, or peptides/proteins of the present invention.

[0021] The present invention provides isolated peptide and protein molecules that consist of, consist essentially of, or comprise the amino acid sequences of the enzyme peptides disclosed in the FIG. 2, (encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3, genomic sequence), as well as all obvious variants of these peptides that are within the art to make and use. Some of these variants are described in detail below.

[0022] As used herein, a peptide is said to be “isolated” or “purified” when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components (the features of an isolated nucleic acid molecule is discussed below).

[0023] In some uses, “substantially free of cellular material” includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins. When the peptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation.

[0024] The language “substantially free of chemical precursors or other chemicals” includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the enzyme peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.

[0025] The isolated enzyme peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. Experimental data as provided in FIG. 1 indicates expression in humans in placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, bladder, and liver. For example, a nucleic acid molecule encoding the enzyme peptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Many of these techniques are described in detail below.

[0026] Accordingly, the present invention provides proteins that consist of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence of such a protein is provided in FIG. 2. A protein consists of an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein.

[0027] The present invention further provides proteins that consist essentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein.

[0028] The present invention further provides proteins that comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. The preferred classes of proteins that are comprised of the enzyme peptides of the present invention are the naturally occurring mature proteins. A brief description of how various types of these proteins can be madelisolated is provided below.

[0029] The enzyme peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins. Such chimeric and fusion proteins comprise a enzyme peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the enzyme peptide. “Operatively linked” indicates that the enzyme peptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the enzyme peptide.

[0030] In some uses, the fusion protein does not affect the activity of the enzyme peptide per se. For example, the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant enzyme peptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence.

[0031] A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al., Current Protocols in Molecular Biology, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A enzyme peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the enzyme peptide.

[0032] As mentioned above, the present invention also provides and enables obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.

[0033] Such variants can readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the enzyme peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the orthologs.

[0034] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0035] The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0036] The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0037] Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the enzyme peptides of the present invention as well as being encoded by the same genetic locus as the enzyme peptide provided herein. The gene encoding the novel enzyme of the present invention is located on a genome component that has been mapped to human chromosome 8 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

[0038] Allelic variants of a enzyme peptide can readily be identified as being a human protein having a high degree (significant) of sequence homology/identity to at least a portion of the enzyme peptide as well as being encoded by the same genetic locus as the enzyme peptide provided herein. Genetic locus can readily be determined based on the genomic information provided in FIG. 3, such as the genomic sequence mapped to the reference human. The gene encoding the novel enzyme of the present invention is located on a genome component that has been mapped to human chromosome 8 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. As used herein, two proteins (or a region of the proteins) have significant homology when the amino acid sequences are typically at least about 70-80%, 80-90%, and more typically at least about 90-95% or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to a enzyme peptide encoding nucleic acid molecule under stringent conditions as more fully described below.

[0039]FIG. 3 provides information on SNPs that have been found in the gene encoding the enzyme of the present invention. SNPs were identified at 55 different nucleotide positions, including a non-synonymous coding SNP at nucleotide position 8031 (protein position 45). The change in the amino acid sequence caused by this SNP is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. Some of these SNPs that are located outside the ORF and in introns may affect gene transcription.

[0040] Paralogs of a enzyme peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the enzyme peptide, as being encoded by a gene from humans, and as having similar activity or function. Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain. Such paralogs will be encoded by a nucleic acid sequence that will hybridize to a enzyme peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below.

[0041] Orthologs of a enzyme peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the enzyme peptide as well as being encoded by a gene from another organism. Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to a enzyme peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins.

[0042] Non-naturally occurring variants of the enzyme peptides of the present invention can readily be generated using recombinant techniques. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the enzyme peptide. For example, one class of substitutions are conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a enzyme peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

[0043] Variant enzyme peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind substrate, ability to phosphorylate substrate, ability to mediate signaling, etc. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. FIG. 2 provides the result of protein analysis and can be used to identify critical domains/regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.

[0044] Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

[0045] Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085 (1989)), particularly using the results provided in FIG. 2. The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as enzyme activity or in assays such as an in vitro proliferative activity. Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).

[0046] The present invention further provides fragments of the enzyme peptides, in addition to proteins and peptides that comprise and consist of such fragments, particularly those comprising the residues identified in FIG. 2. The fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed publicly prior to the present invention.

[0047] As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from a enzyme peptide. Such fragments can be chosen based on the ability to retain one or more of the biological activities of the enzyme peptide or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen. Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length. Such fragments will typically comprise a domain or motif of the enzyme peptide, e.g., active site, a transmembrane domain or a substrate-binding domain. Further, possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures. Predicted domains and functional sites are readily identifiable by computer programs well known and readily available to those of skill in the art (e.g., PROSITE analysis). The results of one such analysis are provided in FIG. 2.

[0048] Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in enzyme peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art (some of these features are identified in FIG. 2).

[0049] Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0050] Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62 (1992)).

[0051] Accordingly, the enzyme peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature enzyme peptide is fused with another compound, such as a compound to increase the half-life of the enzyme peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature enzyme peptide, such as a leader or secretory sequence or a sequence for purification of the mature enzyme peptide or a pro-protein sequence.

[0052] Protein/Peptide Uses

[0053] The proteins of the present invention can be used in substantial and specific assays related to the functional information provided in the Figures; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Where the protein binds or potentially binds to another protein or ligand (such as, for example, in a enzyme-effector protein interaction or enzyme-ligand interaction), the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any or all of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products.

[0054] Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0055] The potential uses of the peptides of the present invention are based primarily on the source of the protein as well as the class/action of the protein. For example, enzymes isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the enzyme. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in the placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, and bladder, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in the liver. A large percentage of pharmaceutical agents are being developed that modulate the activity of enzyme proteins, particularly members of the synthase subfamily (see Background of the Invention). The structural and functional information provided in the Background and Figures provide specific and substantial uses for the molecules of the present invention, particularly in combination with the expression information provided in FIG. 1. Experimental data as provided in FIG. 1 indicates expression in humans in placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, bladder, and liver. Such uses can readily be determined using the information provided herein, that which is known in the art, and routine experimentation.

[0056] The proteins of the present invention (including variants and fragments that may have been disclosed prior to the present invention) are useful for biological assays related to enzymes that are related to members of the synthase subfamily. Such assays involve any of the known enzyme functions or activities or properties useful for diagnosis and treatment of enzyme-related conditions that are specific for the subfamily of enzymes that the one of the present invention belongs to, particularly in cells and tissues that express the enzyme. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in the placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, and bladder, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in the liver.

[0057] The proteins of the present invention are also useful in drug screening assays, in cell-based or cell-free systems. Cell-based systems can be native, i.e., cells that normally express the enzyme, as a biopsy or expanded in cell culture. Experimental data as provided in FIG. 1 indicates expression in humans in placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, bladder, and liver. In an alternate embodiment, cell-based assays involve recombinant host cells expressing the enzyme protein.

[0058] The polypeptides can be used to identify compounds that modulate enzyme activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the enzyme. Both the enzymes of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the enzyme. These compounds can be further screened against a functional enzyme to determine the effect of the compound on the enzyme activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the enzyme to a desired degree.

[0059] Further, the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the enzyme protein and a molecule that normally interacts with the enzyme protein, e.g. a substrate or a component of the signal pathway that the enzyme protein normally interacts (for example, another enzyme). Such assays typically include the steps of combining the enzyme protein with a candidate compound under conditions that allow the enzyme protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the enzyme protein and the target, such as any of the associated effects of signal transduction such as protein phosphorylation, cAMP turnover, and adenylate cyclase activation, etc.

[0060] Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab′)₂, Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).

[0061] One candidate compound is a soluble fragment of the receptor that competes for substrate binding. Other candidate compounds include mutant enzymes or appropriate fragments containing mutations that affect enzyme function and thus compete for substrate. Accordingly, a fragment that competes for substrate, for example with a higher affinity, or a fragment that binds substrate but does not allow release, is encompassed by the invention.

[0062] The invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) enzyme activity. The assays typically involve an assay of events in the signal transduction pathway that indicate enzyme activity. Thus, the phosphorylation of a substrate, activation of a protein, a change in the expression of genes that are up- or down-regulated in response to the enzyme protein dependent signal cascade can be assayed.

[0063] Any of the biological or biochemical functions mediated by the enzyme can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly FIG. 2. Specifically, a biological function of a cell or tissues that expresses the enzyme can be assayed. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in the placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, and bladder, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in the liver.

[0064] Binding and/or activating compounds can also be screened by using chimeric enzyme proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions. For example, a substrate-binding region can be used that interacts with a different substrate then that which is recognized by the native enzyme. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in other than the specific host cell from which the enzyme is derived.

[0065] The proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the enzyme (e.g. binding partners and/or ligands). Thus, a compound is exposed to a enzyme polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Soluble enzyme polypeptide is also added to the mixture. If the test compound interacts with the soluble enzyme polypeptide, it decreases the amount of complex formed or activity from the enzyme target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the enzyme. Thus, the soluble polypeptide that competes with the target enzyme region is designed to contain peptide sequences corresponding to the region of interest.

[0066] To perform cell free drug screening assays, it is sometimes desirable to immobilize either the enzyme protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.

[0067] Techniques for immobilizing proteins on matrices can be used in the drug screening assays. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of enzyme-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques. For example, either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation. Preparations of a enzyme-binding protein and a candidate compound are incubated in the enzyme protein-presenting wells and the amount of complex trapped in the well can be quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the enzyme protein target molecule, or which are reactive with enzyme protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.

[0068] Agents that modulate one of the enzymes of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.

[0069] Modulators of enzyme protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the enzyme pathway, by treating cells or tissues that express the enzyme. Experimental data as provided in FIG. 1 indicates expression in humans in placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, bladder, and liver. These methods of treatment include the steps of administering a modulator of enzyme activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein.

[0070] In yet another aspect of the invention, the enzyme proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with the enzyme and are involved in enzyme activity. Such enzyme-binding proteins are also likely to be involved in the propagation of signals by the enzyme proteins or enzyme targets as, for example, downstream elements of a enzyme-mediated signaling pathway. Alternatively, such enzyme-binding proteins are likely to be enzyme inhibitors.

[0071] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a enzyme protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a enzyme-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the enzyme protein.

[0072] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a enzyme-modulating agent, an antisense enzyme nucleic acid molecule, a enzyme-specific antibody, or a enzyme-binding partner) can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

[0073] The enzyme proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. Experimental data as provided in FIG. 1 indicates expression in humans in placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, bladder, and liver. The method involves contacting a biological sample with a compound capable of interacting with the enzyme protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

[0074] One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein. A biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.

[0075] The peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs. Thus, the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered enzyme activity in cell-based or cell-free assay, alteration in substrate or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

[0076] In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent. Alternatively, the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.

[0077] The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism. Thus, the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes effects both the intensity and duration of drug action. Thus, the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype. The discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages. Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the enzyme protein in which one or more of the enzyme functions in one population is different from those in another population. The peptides thus allow a target to ascertain a genetic predisposition that can affect treatment modality. Thus, in a ligand-based treatment, polymorphism may give rise to amino terminal extracellular domains and/or other substrate-binding regions that are more or less active in substrate binding, and enzyme activation. Accordingly, substrate dosage would necessarily be modified to maximize the therapeutic effect within a given population containing a polymorphism. As an alternative to genotyping, specific polymorphic peptides could be identified.

[0078] The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Experimental data as provided in FIG. 1 indicates expression in humans in placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, bladder, and liver. Accordingly, methods for treatment include the use of the enzyme protein or fragments.

[0079] Antibodies

[0080] The invention also provides antibodies that selectively bind to one of the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof. As used herein, an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross-reactivity.

[0081] As used herein, an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge. The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0082] Many methods are known for generating and/or identifying antibodies to a given target peptide. Several such methods are described by Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0083] In general, to generate antibodies, an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse. The full-length protein, an antigenic peptide fragment or a fusion protein can be used. Particularly important fragments are those covering functional domains, such as the domains identified in FIG. 2, and domain of sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures.

[0084] Antibodies are preferably prepared from regions or discrete fragments of the enzyme proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or enzyme/binding partner interaction. FIG. 2 can be used to identify particularly important regions while sequence alignment can be used to identify conserved and unique sequence fragments.

[0085] An antigenic fragment will typically comprise at least 8 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues. Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness (see FIG. 2).

[0086] Detection on an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ₁₂₅I, ¹³¹I, ³⁵S or ³H.

[0087] Antibody Uses

[0088] The antibodies can be used to isolate one of the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells. In addition, such antibodies are useful to detect the presence of one of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in the placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, and bladder, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in the liver. Further, such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full length protein can be used to identify turnover.

[0089] Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form, the antibody can be prepared against the normal protein. Experimental data as provided in FIG. 1 indicates expression in humans in placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, bladder, and liver. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein.

[0090] The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism. Experimental data as provided in FIG. 1 indicates expression in humans in placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, bladder, and liver. The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.

[0091] Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities. The antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.

[0092] The antibodies are also useful for tissue typing. Experimental data as provided in FIG. 1 indicates expression in humans in placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, bladder, and liver. Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.

[0093] The antibodies are also useful for inhibiting protein function, for example, blocking the binding of the enzyme peptide to a binding partner such as a substrate. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane. See FIG. 2 for structural information relating to the proteins of the present invention.

[0094] The invention also encompasses kits for using antibodies to detect the presence of a protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use. Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nuleic acid arrays and similar methods have been developed for antibody arrays.

[0095] Nucleic Acid Molecules

[0096] The present invention further provides isolated nucleic acid molecules that encode a enzyme peptide or protein of the present invention (cDNA, transcript and genomic sequence). Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the enzyme peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof.

[0097] As used herein, an “isolated” nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence. The important point is that the nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.

[0098] Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.

[0099] For example, recombinant DNA molecules contained in a vector are considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

[0100] Accordingly, the present invention provides nucleic acid molecules that consist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.

[0101] The present invention further provides nucleic acid molecules that consist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.

[0102] The present invention further provides nucleic acid molecules that comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences. Such a nucleic acid molecule can have a few additional nucleotides or can comprises several hundred or more additional nucleotides. A brief description of how various types of these nucleic acid molecules can be readily made/isolated is provided below.

[0103] In FIGS. 1 and 3, both coding and non-coding sequences are provided. Because of the source of the present invention, humans genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acid molecules in the Figures will contain genomic intronic sequences, 5′ and 3′ non-coding sequences, gene regulatory regions and non-coding intergenic sequences. In general such sequence features are either noted in FIGS. 1 and 3 or can readily be identified using computational tools known in the art. As discussed below, some of the non-coding regions, particularly gene regulatory elements such as promoters, are useful for a variety of purposes, e.g. control of heterologous gene expression, target for identifying gene activity modulating compounds, and are particularly claimed as fragments of the genomic sequence provided herein.

[0104] The isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.

[0105] As mentioned above, the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the enzyme peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre- pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5′ and 3′ sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.

[0106] Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).

[0107] The invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the enzyme proteins of the present invention that are described above. Such nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.

[0108] The present invention further provides non-coding fragments of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents. A promoter can readily be identified as being 5′ to the ATG start site in the genomic sequence provided in FIG. 3.

[0109] A fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene.

[0110] A probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.

[0111] Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence shown in the Figure sheets or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown in the Figure sheets or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene. The gene encoding the novel enzyme of the present invention is located on a genome component that has been mapped to human chromosome 8 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

[0112]FIG. 3 provides information on SNPs that have been found in the gene encoding the enzyme of the present invention. SNPs were identified at 55 different nucleotide positions, including a non-synonymous coding SNP at nucleotide position 8031 (protein position 45). The change in the amino acid sequence caused by this SNP is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. Some of these SNPs that are located outside the ORF and in introns may affect gene transcription.

[0113] As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to low stringency hybridization conditions are well known in the art.

[0114] Nucleic Acid Molecule Uses

[0115] The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptide described in FIG. 2 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in FIG. 2. As illustrated in FIG. 3, SNPs were identified at 55 different nucleotide positions.

[0116] The probe can correspond to any sequence along the entire length of the nucleic acid molecules provided in the Figures. Accordingly, it could be derived from 5′ noncoding regions, the coding region, and 3′ noncoding regions. However, as discussed, fragments are not to be construed as encompassing fragments disclosed prior to the present invention.

[0117] The nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence.

[0118] The nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the peptide sequences. Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in situ expression of a gene and/or gene product. For example, an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.

[0119] The nucleic acid molecules are also useful for expressing antigenic portions of the proteins.

[0120] The nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods. The gene encoding the novel enzyme of the present invention is located on a genome component that has been mapped to human chromosome 8 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

[0121] The nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention.

[0122] The nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.

[0123] The nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.

[0124] The nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides.

[0125] The nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides.

[0126] The nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distribution of nucleic acid expression. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in the placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, and bladder, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in the liver. Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, tissue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in enzyme protein expression relative to normal results.

[0127] In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA includes Southern hybridizations and in situ hybridization.

[0128] Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a enzyme protein, such as by measuring a level of a enzyme-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a enzyme gene has been mutated. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in the placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, and bladder, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in the liver.

[0129] Nucleic acid expression assays are useful for drug screening to identify compounds that modulate enzyme nucleic acid expression.

[0130] The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the enzyme gene, particularly biological and pathological processes that are mediated by the enzyme in cells and tissues that express it. Experimental data as provided in FIG. 1 indicates expression in humans in placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, bladder, and liver. The method typically includes assaying the ability of the compound to modulate the expression of the enzyme nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired enzyme nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the enzyme nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.

[0131] The assay for enzyme nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the enzyme protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.

[0132] Thus, modulators of enzyme gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined. The level of expression of enzyme mRNA in the presence of the candidate compound is compared to the level of expression of enzyme mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.

[0133] The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate enzyme nucleic acid expression in cells and tissues that express the enzyme. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in the placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, and bladder, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in the liver. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.

[0134] Alternatively, a modulator for enzyme nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the enzyme nucleic acid expression in the cells and tissues that express the protein. Experimental data as provided in FIG. 1 indicates expression in humans in placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, bladder, and liver.

[0135] The nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the enzyme gene in clinical trials or in a treatment regimen. Thus, the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.

[0136] The nucleic acid molecules are also useful in diagnostic assays for qualitative changes in enzyme nucleic acid expression, and particularly in qualitative changes that lead to pathology. The nucleic acid molecules can be used to detect mutations in enzyme genes and gene expression products such as mRNA. The nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the enzyme gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the enzyme gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a enzyme protein.

[0137] Individuals carrying mutations in the enzyme gene can be detected at the nucleic acid level by a variety of techniques. FIG. 3 provides information on SNPs that have been found in the gene encoding the enzyme of the present invention. SNPs were identified at 55 different nucleotide positions, including a non-synonymous coding SNP at nucleotide position 8031 (protein position 45). The change in the amino acid sequence caused by this SNP is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. Some of these SNPs that are located outside the ORF and in introns may affect gene transcription. The gene encoding the novel enzyme of the present invention is located on a genome component that has been mapped to human chromosome 8 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.

[0138] Alternatively, mutations in a enzyme gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.

[0139] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature.

[0140] Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and S1 protection or the chemical cleavage method. Furthermore, sequence differences between a mutant enzyme gene and a wild-type gene can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).

[0141] Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers et al., Nature 313:495 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.

[0142] The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the enzyme gene in an individual in order to select an appropriate compound or dosage regimen for treatment. FIG. 3 provides information on SNPs that have been found in the gene encoding the enzyme of the present invention. SNPs were identified at 55 different nucleotide positions, including a non-synonymous coding SNP at nucleotide position 8031 (protein position 45). The change in the amino acid sequence caused by this SNP is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. Some of these SNPs that are located outside the ORF and in introns may affect gene transcription.

[0143] Thus nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.

[0144] The nucleic acid molecules are thus useful as antisense constructs to control enzyme gene expression in cells, tissues, and organisms. A DNA antisense nucleic acid molecule is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of enzyme protein. An antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and thus block translation of mRNA into enzyme protein.

[0145] Alternatively, a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of enzyme nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired enzyme nucleic acid expression. This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the enzyme protein, such as substrate binding.

[0146] The nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in enzyme gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired enzyme protein to treat the individual.

[0147] The invention also encompasses kits for detecting the presence of a enzyme nucleic acid in a biological sample. Experimental data as provided in FIG. 1 indicates that the enzymes of the present invention are expressed in humans in the placenta, T-cells from T-cell leukemia, fetal brain, pancreas, Burkitt lymphoma, and bladder, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in the liver. For example, the kit can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting enzyme nucleic acid in a biological sample; means for determining the amount of enzyme nucleic acid in the sample; and means for comparing the amount of enzyme nucleic acid in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect enzyme protein mRNA or DNA.

[0148] Nucleic Acid Arrays

[0149] The present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

[0150] As used herein “Arrays” or “Microarrays” refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support. In one embodiment, the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et al., U.S. Pat. No. 5,807,522.

[0151] The microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5′, or 3′, sequence, sequential oligonucleotides which cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.

[0152] In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest (or an ORF identified from the contigs of the present invention) is typically examined using a computer algorithm which starts at the 5′ or at the 3′ end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The “pairs” will be identical, except for one nucleotide that preferably is located in the center of the sequence. The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from two to one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.

[0153] In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a “gridded” array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation.

[0154] In order to conduct sample analysis using a microarray or detection kit, the RNA or DNA from a biological sample is made into hybridization probes. The mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit. The biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.

[0155] Using such arrays, the present invention provides methods to identify the expression of the enzyme proteins/peptides of the present invention. In detail, such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample. Such assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the enzyme gene of the present invention. FIG. 3 provides information on SNPs that have been found in the gene encoding the enzyme of the present invention. SNPs were identified at 55 different nucleotide positions, including a non-synonymous coding SNP at nucleotide position 8031 (protein position 45). The change in the amino acid sequence caused by this SNP is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. Some of these SNPs that are located outside the ORF and in introns may affect gene transcription.

[0156] Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the novel fragments of the Human genome disclosed herein. Examples of such assays can be found in Chard, T, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0157] The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized.

[0158] In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.

[0159] Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the Human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.

[0160] In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe. One skilled in the art will readily recognize that the previously unidentified enzyme gene of the present invention can be routinely identified using the sequence information disclosed herein can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays.

[0161] Vectors/Host Cells

[0162] The invention also provides vectors containing the nucleic acid molecules described herein. The term “vector” refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules. When the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0163] A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules. Alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.

[0164] The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules. The vectors can function in prokaryotic or eukaryotic cells or in both (shuttle vectors).

[0165] Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell. The nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.

[0166] The regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.

[0167] In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0168] In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0169] A variety of expression vectors can be used to express a nucleic acid molecule. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0170] The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.

[0171] The nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.

[0172] The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

[0173] As described herein, it may be desirable to express the peptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the peptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enteroenzyme. Typical fusion expression vectors include pGEX (Smith et al., Gene 67:3140 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).

[0174] Recombinant protein expression can be maximized in host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)119-128). Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli. (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0175] The nucleic acid molecules can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).

[0176] The nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et. al., Virology 170:31-39 (1989)).

[0177] In certain embodiments of the invention, the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)).

[0178] The expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0179] The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).

[0180] The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.

[0181] The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0182] Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the nucleic acid molecule vector.

[0183] In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.

[0184] Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.

[0185] While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell-free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.

[0186] Where secretion of the peptide is desired, which is difficult to achieve with multi-transmembrane domain containing proteins such as enzymes, appropriate secretion signals are incorporated into the vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides.

[0187] Where the peptide is not secreted into the medium, which is typically the case with enzymes, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The peptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.

[0188] It is also understood that depending upon the host cell in recombinant production of the peptides described herein, the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.

[0189] Uses of Vectors and Host Cells

[0190] The recombinant host cells expressing the peptides described herein have a variety of uses. First, the cells are useful for producing a enzyme protein or peptide that can be further purified to produce desired amounts of enzyme protein or fragments. Thus, host cells containing expression vectors are useful for peptide production.

[0191] Host cells are also useful for conducting cell-based assays involving the enzyme protein or enzyme protein fragments, such as those described above as well as other formats known in the art. Thus, a recombinant host cell expressing a native enzyme protein is useful for assaying compounds that stimulate or inhibit enzyme protein function.

[0192] Host cells are also useful for identifying enzyme protein mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant enzyme protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native enzyme protein.

[0193] Genetically engineered host cells can be further used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a enzyme protein and identifying and evaluating modulators of enzyme protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.

[0194] A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the enzyme protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.

[0195] Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the enzyme protein to particular cells.

[0196] Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.

[0197] In another embodiment, transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992). Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

[0198] Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. Nature 385:810-813 (1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G_(o) phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring born of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

[0199] Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect substrate binding, enzyme protein activation, and signal transduction, may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo enzyme protein function, including substrate interaction, the effect of specific mutant enzyme proteins on enzyme protein function and substrate interaction, and the effect of chimeric enzyme proteins. It is also possible to assess the effect of null mutations, that is, mutations that substantially or completely eliminate one or more enzyme protein functions.

[0200] All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

1 6 1 1606 DNA Human 1 gcgcctgggg accgcagagg tgagagtcgc gcccgggagt ccgccgcctg cgccaggatg 60 gagttcgtga aatgccttgg ccaccccgaa gagttctaca acctggtgcg cttccggatc 120 gggggcaagc ggaaggtgat gcccaagatg gaccaggact cgctcagcag cagcctgaaa 180 acttgctaca agtatctcaa tcagaccagt cgcagtttcg cagctgttat ccaggcgctg 240 gatggggaaa tgcgcaacgc agtgtgcata ttttatctgg ttctccgagc tctggacaca 300 ctggaagatg acatgaccat cagtgtggaa aagaaggtcc cgctgttaca caactttcac 360 tctttccttt accaaccaga ctggcggttc atggagagca aggagaagga tcgccaggtg 420 ctggaggact tcccaacgta ctgccactat gttgctgggc tggtcggaat tggcctttcc 480 cgtcttttct cagcctcaga gtttgaagac cccttagttg gtgaagatac agaacgtgcc 540 aactctatgg gcctgtttct gcagaaaaca aacatcatcc gtgactatct ggaagaccag 600 caaggaggaa gagagttctg gcctcaagag gtttggagca ggtatgttaa gaagttaggg 660 gattttgcta agccggagaa tattgacttg gccgtgcagt gcctgaatga acttataacc 720 aatgcactgc accacatccc agatgtcatc acctaccttt cgagactcag aaaccagagt 780 gtgtttaact tctgtgctat tccacaggtg atggccattg ccactttggc tgcctgttat 840 aataaccagc aggtgttcaa aggggcagtg aagattcgga aagggcaagc agtgaccctc 900 atgatggatg ccaccaatat gccagctgtc aaagccatca tatatcagta tatggaagag 960 atttatcata gaatccccga ctcagaccca tcttctagca aaacaaggca gatcatctcc 1020 accatccgga cgcagaatct tcccaactgt cagctgattt cccgaagcca ctactccccc 1080 atctacctgt cgtttgtcat gcttttggct gccctgagct ggcagtacct gaccactctc 1140 tcccaggtaa cagaagacta tgttcagact ggagaacact gatcccaaat ttgtccatag 1200 ctgaagtcca ccataaagtg gatttacttt ttttctttaa ggatggatgt tgtgttctct 1260 ttattttttt cctactactt taatccctaa aagaacgctg tgtggctggg acctttagga 1320 aagtgaaatg caggtgagaa gaacctaaac atgaaaggaa agggtgcctc atcccagcaa 1380 cctgtccttg tgggtgatga tcactgtgct gcttgcggct catggcagag cattcagtgc 1440 cacggtttag gtgaagtcgc tgcatatgtg actgtcatga gatcctactt agtatgatcc 1500 tggctagaat gataattaaa agtatttaat ttgaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 1606 2 374 PRT Human 2 Met Glu Phe Val Lys Cys Leu Gly His Pro Glu Glu Phe Tyr Asn Leu 1 5 10 15 Val Arg Phe Arg Ile Gly Gly Lys Arg Lys Val Met Pro Lys Met Asp 20 25 30 Gln Asp Ser Leu Ser Ser Ser Leu Lys Thr Cys Tyr Lys Tyr Leu Asn 35 40 45 Gln Thr Ser Arg Ser Phe Ala Ala Val Ile Gln Ala Leu Asp Gly Glu 50 55 60 Met Arg Asn Ala Val Cys Ile Phe Tyr Leu Val Leu Arg Ala Leu Asp 65 70 75 80 Thr Leu Glu Asp Asp Met Thr Ile Ser Val Glu Lys Lys Val Pro Leu 85 90 95 Leu His Asn Phe His Ser Phe Leu Tyr Gln Pro Asp Trp Arg Phe Met 100 105 110 Glu Ser Lys Glu Lys Asp Arg Gln Val Leu Glu Asp Phe Pro Thr Tyr 115 120 125 Cys His Tyr Val Ala Gly Leu Val Gly Ile Gly Leu Ser Arg Leu Phe 130 135 140 Ser Ala Ser Glu Phe Glu Asp Pro Leu Val Gly Glu Asp Thr Glu Arg 145 150 155 160 Ala Asn Ser Met Gly Leu Phe Leu Gln Lys Thr Asn Ile Ile Arg Asp 165 170 175 Tyr Leu Glu Asp Gln Gln Gly Gly Arg Glu Phe Trp Pro Gln Glu Val 180 185 190 Trp Ser Arg Tyr Val Lys Lys Leu Gly Asp Phe Ala Lys Pro Glu Asn 195 200 205 Ile Asp Leu Ala Val Gln Cys Leu Asn Glu Leu Ile Thr Asn Ala Leu 210 215 220 His His Ile Pro Asp Val Ile Thr Tyr Leu Ser Arg Leu Arg Asn Gln 225 230 235 240 Ser Val Phe Asn Phe Cys Ala Ile Pro Gln Val Met Ala Ile Ala Thr 245 250 255 Leu Ala Ala Cys Tyr Asn Asn Gln Gln Val Phe Lys Gly Ala Val Lys 260 265 270 Ile Arg Lys Gly Gln Ala Val Thr Leu Met Met Asp Ala Thr Asn Met 275 280 285 Pro Ala Val Lys Ala Ile Ile Tyr Gln Tyr Met Glu Glu Ile Tyr His 290 295 300 Arg Ile Pro Asp Ser Asp Pro Ser Ser Ser Lys Thr Arg Gln Ile Ile 305 310 315 320 Ser Thr Ile Arg Thr Gln Asn Leu Pro Asn Cys Gln Leu Ile Ser Arg 325 330 335 Ser His Tyr Ser Pro Ile Tyr Leu Ser Phe Val Met Leu Leu Ala Ala 340 345 350 Leu Ser Trp Gln Tyr Leu Thr Thr Leu Ser Gln Val Thr Glu Asp Tyr 355 360 365 Val Gln Thr Gly Glu His 370 3 40090 DNA Human misc_feature (1)...(40090) n = A,T,C or G 3 tatttattcc taattaaatg gggaggaaag tctttgaaga ggaacctcta ctttactttt 60 tataccgtca tggctggaaa ctaagttttt aagatttttc tggggttccc ttggccgagg 120 tggggagtgg gagggctgtc cagtggtagg gacttaggat ttttagttta cagtagtagg 180 ggaaacactc tgtaatctaa tacataagta aatgatgtat tagaatatgg taaatatagg 240 caagtagacc cccactggga ttagcagtgg tggaaatgtg agagagggca aacaggtggg 300 tctagatgag gtgtgagcag actcgagggg cacaggagtt agtcaagcca gtatctgggg 360 gatagtgcag gaatagtgaa cagctagaca aaaagtccta gggccagaga aagcaaaagc 420 ataagagatg gaggccagag aggtaatctg ggtggaaggc tgcagcctct caggatccct 480 ataggtgctt tggcttttgt tggagagaca ctgaacagct ttgggcagtg aacgtacctg 540 acaggtttcc tgtttgtttt tgagatgaag tctcgctctt gtcccccagg ctggagtgca 600 atagcgcgat ctcagctcac tgcaacctct gcctcctgtg ttcaagcgat tctcctgcct 660 cagcctccca ggtagctggg attataggcg cctgccacca tgcctggcta atttttgtat 720 ttttagtaga gacgcagttt cagcatgttg gccaggctgg tcttgaactc cagacctcag 780 gtgatccgcc cgccttggcc tcccaaagtg ctgggattac aggcgtgagc caccgcgctc 840 ggctagacct gacaggtttt aaaaggatta ctggttgctg tgttaaaaca gactgcagga 900 tggcttaggt agccagtagg tttttttttt ttttggagac gtagtcttgc tctgttggcc 960 tggctggagt gcagcggtgt catcttggct cactgcaaac tccgcttccc gggttcaagt 1020 gattctcctg cctcagcctc cggagtagtt gggactacag gcgcccacca ccacactcgg 1080 cttttttgta tttttagtag agacgggttt caccatgttg gccaggatgg tctcgatctc 1140 ttgacctcgt gatccacccg ccttggcctc ccaaagtgtt gcgattacag gcgtgagcca 1200 ccacgcctgg acgggtagcc agtagtttct agggctggag agatctagga tgagagaagt 1260 ttccacattc ctgttacagg ctctctaagg cttcagctcc tttttctagg actaagctgg 1320 atctcaagta aacactagag agggggcagc tgaagctcca ggagtgtgtg gggctccctg 1380 gggctggatg gcggtggcgg gcaggcgagc tgggctgtgc tcgggtgtgt tacagtaaag 1440 acgcccagct tggcgctggc ccggcctttt cacggtttta ggctctacag agagcggctg 1500 cagagctcac ccggctggca ggagccaccg aggccggaca cgtgggcgac ttattgacca 1560 agtggggagg aagcagcccc gcactgctct cccgactgcg gaccaccgtt gggctcatgc 1620 gcatcataag ccccaccgcc tcacctccag tccccacagc gttcgcgctc ccagccgggg 1680 taagcggaag aaaacaaagg cccggctcca tcagggcacc aatcccgctc gtcggcctct 1740 ttctcggcct ccaatgagct tctagggtgt tatcacgcca gtctccttcc gcgactgatt 1800 ggccggggtc ttcctagtgt gagcggccct ggccaatcag gcgcccgtca gcccacccca 1860 cgaggccgca gctagccccg ctggcggccg aggccggttg aagtgggcgg agcggcgggc 1920 ggggcgtcgc cgtactaggc ctgccccctg tccggccagc ccctcgaagc acctactcca 1980 caggtccagc cggccggtga gcgcctgggg accgcagagg tgagagtcgc gcccgggagt 2040 ccgccgcctg cgccaggatg gagttcgtga aatgccttgg ccaccccgaa gagttctaca 2100 acctggtgcg cttccggatc gggggcaagc ggaaggtgat gcccaagatg gaccaggtgg 2160 gccgagcctc cctgcttgcc cggggcgggg aaggagctcg ctgggccggc ctcagggcct 2220 gagcggccgg gcccggatct ggggcaaggg gcgcggcgag cagggccgac gcctgggtgt 2280 tcccgtcccc ctttcctcga gccttccccc tgtagggccc gggtggacgc ggccgtcctg 2340 gctgacctgt ccctgccccc gcaagccgcc ctgggcatga gcgacttttg cgtggttccc 2400 ggtggttgcg ctccccgttt cgtcccctcc gtgagcatcg gcgcttaccg gtattttaac 2460 ccgagggtta cacatctgag gcaatgtggg tgggttacgc gggagaggac gagtgagttt 2520 tttggtaagc ggaatgaact atgcagataa catcacatga aggccgtttc tggaatgaag 2580 tctgactcct ccagtttcac cacctcttcc ggagctctcc ccgccttgct gccttccatc 2640 gcttcatcct cggtgcttcc tgagttttaa aatcgcctat ctacgcttcc aagttccaat 2700 gagttatcta acgtctatgg attagctagg tggttggtgg aaggtcagaa cttggtttta 2760 cttagatttt tatctgcctc atgcctgtac tatttgttta atgaatgcat aggaggtgtt 2820 tttattccaa caagaaaatt attcgtacgc gattattgaa tgaatagaca aattcagcca 2880 agttcttctg gtctggacca gcctggctga tttctgtaac ttttttgggc caacaggaca 2940 gtagcaaatg tgactcaggc cgaggcttga taggtgcctg aacatcggag tctttctttc 3000 agtgtccatg tgcttcagta aacacactag aaaataaatt tctggttttt gtccccagta 3060 gactacaccc tcatttggtg ttatttttca cgtgctatct ttaatacagg tacatccttc 3120 agtctatttg tagaacattc agttttcttc atcttttctt tgccggtgct acattatttg 3180 aattattttg ctacagaata acttctatta tttgatatgg cagatgtcac tttttatatt 3240 tagatatagc attcatttat ttaacaaata tttgacgacc agttgtatat cagatagtgt 3300 tctaggtgct ggaggtacaa cagtgaacaa gctaggtgaa gaccttgatt ttataaaact 3360 tactttttag tggaagagag acaatttaaa aaagcgaatg tacagttttt cacgtggaga 3420 aaagcactgc agaggaagat actagcaggg caagggatct gagtgcagtc agacctcatt 3480 tgggtccaga cttcattcct ctatgtctct ttcctttcta cagaaagact gttagagaaa 3540 atggtagcat tggtttcctg ttgggaggga aagtgggtgg tcatggtaag tgggtagaga 3600 aagacttcac agtatactgt ttttgtacat tttgagtttt tttaaaagcg agacttgagc 3660 tattctagct ctgataatat ggtgcagtat ttgttatgtt agttgtagtc tttctgggca 3720 gtttttacat ccccatgagc cgttaaaaaa atacctgaac ctttaattag gggaaataaa 3780 ttggaaaaat acatttccct tcacttaaca ttatcttagt ttctcttttt tttttttttt 3840 ttttttgaga tggagtcttg ctctgttacc caggctggag tgcagtggtg gcgggacctc 3900 agctagatgc agcctccgcc tcctgggttc aagcaattct cctgcctcag cctgctgagt 3960 agctgggatt acaggcacct gccactacgc ccggctgatt ttttggtatt tttagtagag 4020 acggggtttc accatgttgg cgaggctggt tttgaactct tgacctcaag tgatctgctc 4080 gccttggtct cccaaagtgc taggattaca ggcgtgagcc actgcacccg gccttttttt 4140 tttttttttt gagggggggg tctcactcca tcgtccaggc tagaatgctg tggcctgaac 4200 atgactcact ccagttttga cttccttggc tgaagccatc ctcccacctc ggcttcctga 4260 tcccgagtag ctgggactcc aggcacgtgt caccaatgca tggctaattt ttaaattttt 4320 ttgtagacac aatgtctcgc tgcattgccc aggctggtct tgaactcctg agctcaagcg 4380 attttcccac ctcagccttc aaagtgctgg gattacaggt gtgagccact gcacccaacc 4440 agtttctctc tgcaaactag ggaaaaaatt tacgcttagc agatattgag ggctgattat 4500 ttctatcaca gaagcatttg gctatagaat ttcagggttt agtaaacttg atttacactg 4560 aatttttagg tgcatatcag taaatctacg ggcatatgcc gcctgcaagt tgtgtggcat 4620 cacccaaaag ccgagagtaa tggaaagagc aggctgttag taatcaggca gatctggctc 4680 ctgtccaatc taaatcctgt tatttagact aatatcttaa gtctgttatt aagtccgatt 4740 tctgacgcta ttaagttagg tgaacaacct tggtaactta acctctgaac cacagttact 4800 tcatctgtaa aatagggatg tatgtatggt aacgattttt taaccacaac ttcccaactc 4860 taagatggtc tgaaaagaat tttttgagtg tttggctcag aatcacttgg cagcaaaacc 4920 tgacttgaag ttgaggcttc attcatccca cttagtatat tcaaatgttt tgctaaagaa 4980 ataattatga ggtgctactt cacactgact agggttgtat atgcatttta ttgcctattt 5040 tctaaaacac taaaaatgct aaattctgcc ccaggtcttg ccacagatgt ttcagtggac 5100 tatgggcctg tgagacctta aagggttgat tgagtaagga tcacaggtga tgtccgcatt 5160 gtgcttggca tggagttaag tgcttgataa atggtggtta tcaatctgat tatgtaaatt 5220 tatgtaaatt cagttctcaa gtttgtggtt tttttcccct cctggagaaa tctattctat 5280 tttaaagtga ggaaggctcc gtggagggct ggtagctggt agctgttcac ttgtggaact 5340 ttcagcctga ggctggagcc ccttcctggg agtctggtct tgtcgtcttc ctgaccaccc 5400 ccacaccctt cctctaaatt ccctccatcc ctgtttttct cccgcttgcg agcttttggg 5460 agtgtgctga atctcagact gcaatagata aacccaagag ggacaggcac cagtagcctg 5520 agcttgcttt ctcccctggc tcatgggaat caagcagtag aaatttttag tgagtgttgt 5580 tttccatagt atgcttacta gttgtgtctt cctgttttgt tcttggtgat ttgaagaaac 5640 ctgtttacaa ggtaagggac tgaaacaaat aggtgacagg aaaaagagca gcaggggtac 5700 gagctggagg agtaagtggc ttggcttgct ctctttcaga atggagggct gtatggaaag 5760 gaggggtagt gttcttgaag agtgttgggg tttaaatcta gggggaccgt gtcttggcat 5820 tgattgaaac tcctggctta acatcacccc gaaactgtta gttggactga acatgacatt 5880 tggcagtgca gttaaaaaca cttcctgctg tagcctggta atggtcaggc tatgtgaaga 5940 gctgctctgg agctcagtcc agagcgggta ttctgtttct ttcactctga aatcctgcct 6000 ctcgatattt tgagaaggaa ggagttggtg aattgtttta aaatcctcga tgaatgtctt 6060 catttattca tgacaccact tctgaatata tttatgtgcc agacgctgaa gtttactaat 6120 attatggtgc ccagtaaata cttgttttta ctaatatttt ttatggcaat aaaatgactt 6180 tttcaggatt atgtgattta aaagattgac ccttttggca aaatacgtat tcatgatagg 6240 aaatatatac aacatagttc acttaaacct cccaccagag cccagggttc actgttacca 6300 ttctgaagtg actggaattt cctagaagtg gatatgccat atttttttaa ccactcctat 6360 tggatatttg ttttttattt ttttgagatg gggtcccact ctgcagtgta caatatcata 6420 gttcactgta acgtgtatct cttgggctca agcgatcctc cccacctcag cctccctgag 6480 tagctagtct tcagtagcta gactataggt gggcgccacc acagctggct ttttaaaaaa 6540 ttttttatga acacgaggtc tcactatgtt gcccaggctg ccctcaaact cctgggctca 6600 agtgattctc ccaccttggc cttccgaagt gcagggatta taggcgtgcg ccactgcacc 6660 cggccctgtt ggataaatga ttccagtctc tcccaaaaag aactgttgta agactgtggg 6720 gtgaggggag ggaagggaca aataggaacc cgccgtattt tccactccct gtgggcctaa 6780 aactgctcta aaaaatagtc catgaaaaaa tacatagtac aaacagcaac tctttctgat 6840 atgcttgcat ttaaaatcag gctttttctc ccttttggaa aaacacagtc cttgtttgct 6900 ttagggaaga gtaaaggtca gtgcgctgca ttgcattaat ttcgaaggga aagatgagaa 6960 gacatcttga aaggaatggc tggctttcta gagaatagta gaggcttaat aggtgtcata 7020 gaaaaaccag ggttggacag tggtagtaaa acggcaaaac agattttatt cagaaaaact 7080 actgcagtaa gaggagagag acctcggtac agaactgctc cactgcgaat acaaagaaaa 7140 gtaggaattg atggcggggg agccggatgt cagtggatgg aaaattatta cgaggaaaca 7200 caggggtgtg cattcttgct gaaggcaggc cagagttatc agacatcacc tgagggatgg 7260 agggggatgt ggaacctaat cggctgtcta gggtgatcag atactgaagt tgggggattc 7320 tggtcaaatc aatttagcag gattcttggt aaaactgggc gatgcaaaga cagatgcgtt 7380 gagtacaaag tccaggcttt attgggaaga ggatttcagc ggagcccgag tagagtttgg 7440 tctagggaga ctctgtcact gggaggacga gcgagccgct cggaagtgcg ctgggttctc 7500 ttagcggcca gtgggttctg gtgagaaggg caacagcggg aggaggcgcc ggtgcggagc 7560 gggaggccgg gggcggggct gcggggctgc ggggcgggcc cgttgtgggt cggcccagcg 7620 cgtattcgag tagagggcga gcccgtcccg cctctcgtcg ggcgcttccc agatctgctt 7680 gagtctatgg aggaaaaact ccgcggggtc cgcgattccc atggccgcag ccgcctgcgg 7740 caccaaggcc atggccctct tcaagcgcac cttggtgctg agtcccgccg cggcgcccag 7800 gggcccgggc gcaggcaccg ccccgcgggg ctgctgcttg cctcctgccg cctggccctg 7860 caaggactgg cctcggggag agggcggcag gctgtggagc cgcctgcccc agtcccagtc 7920 ccactcccac tcccactccc actcccactc ctgctcctcg acgtctccca ccgccgtgtg 7980 tgttgtctgc ccgcaggact cgctcagcag cagcctgaaa acttgctaca agtatctcaa 8040 tcagaccagt cgcagtttcg cagctgttat ccaggcgctg gatggggaaa tgcggtgagt 8100 gatggaggca gcgcctctgg cttggaggaa agcttgtccg ggacttttga gtgtgttgga 8160 agctaccttt tgatatagcg ctcagcgttg cagcctcgtt gctgtggctt atccagaaca 8220 tagcccggcc ctacgtgttt actttagaaa gcccttccag gctctttgcc atctagtaga 8280 gtccctgcgg gcccagcctt tcagagaagg ggggggaggg ggtgatgttt attaactttt 8340 tttagtcttg gcagctgaac ctgcctgtga gcaggtcgtg tatttctcgg cttcccttat 8400 ccaactttgc atttctattt ctagcatatt gggttgattc ttttgaagct gcctctgtgc 8460 acattacacc catgaactta gaccagttgc ctttatgtat gatcgtattt atactgagaa 8520 gttactgtgt tttttgactt tcttttctat ttgctacata ttagttcggt ctaaacgttt 8580 ggtcttctgg tctccatagt tctacattgg ttaaatgcaa ctcacttctg ggagtagtgg 8640 tgacattcaa ctagtaggct ttttaataaa ctacagaagt tcattactct catgtaagga 8700 aggaaaacta atgtaacttt cgttaagtat gaaaagcgtt ggatatcctt atagttcttt 8760 agagttaagg gtgagatggg tttagaaagt ggccaggcac aagttatttt aaaataaaaa 8820 atctttggct gtttgttcca atatattaat agttttccct tttttacagc aacgcagtgt 8880 gcatatttta tctggttctc cgagctctgg acacactgga agatgacatg accatcagtg 8940 tggaaaagaa ggtcccgctg ttacacaact ttcactcttt cctttaccaa ccagactggc 9000 ggttcatgga gagcaaggag aaggatcgcc aggtgctgga ggacttccca acggtgagtg 9060 gggttacgca tcttgtctac ggactgttgt gttcataatt gctaacgtgg ttgtccggta 9120 gcctccatac atgtggagaa aggttaaata agcattctga gggcagcata atgtgagggt 9180 taaaaactcc ggtagccaag actctgaagc caggctgcct gggttggaat ctcaaatctc 9240 ccacttacta aactgttggt tacttacaaa gactctctgt gcctcagttt cttcatctgt 9300 aaaatagggg taataataac acctacctca tggtattctg aggattcaaa gaattaacgt 9360 aggtaatgct cttagaatgt tagctactgc tgttattatc agtattggaa gtccagtgtt 9420 tcttcctgtg ggaagacgca gtcaaatttt agtgttgtga aagattctca ggctagctca 9480 caaaagcctg ccgactgtat gatgcagcct acctgtaaca ctgctggcct cttgactacc 9540 cggagcctgg tagcatggga ctgctgctca cgatgggcag cagcctggca tgggggcggt 9600 gtctgttggc agctagggcg agcctctgcc acttcacctg tgatcctggg caagttcctt 9660 atctgctttg tgtctccgtc tcctcgtttg taaagttaga gctgagagga ttaatttcgc 9720 acatataaag tacttagtgc ctggtacagg gtaagtattc tgtaagtatt agctatttgg 9780 tctattttgt tggagtaaag tgggttatag ttaaaatcct aagattttta aagtccctca 9840 agttcacgtg gacatctgcc taggtcctac tatcctagaa ttcgcatgtc ttatcacaca 9900 aataactgat tcttccatat cttataaata aaggtttgat ttagcaaagt cacatgttgt 9960 gtaatagctc gaagaagccc tttttgtcca cagttgccag agcttttgga gaacagtcct 10020 tatgttattg aaacaaacct aatctgtagc tgagttggga gggagctaag tggacagaga 10080 gtcctccacc caaacaaaag aatctttgat tcttgggcat aatgggagca atatttaaaa 10140 aaaaaaaaaa aaaaaaaaaa ggaatgtttg gggaagactc ttgcggtgca aaggctgttt 10200 cagattgctg agatcagacc ttaagtacca aagcccaaat atagtacaac ataatacaaa 10260 tgagaagaaa atagctgaag aataattcga gtttatacag tacaattcaa gagaagaaag 10320 aaaatttatg acgactagct gggtgagaat tagaactgta accctgggaa ggtcctggtg 10380 atttgactct cacaggacac ctgatgacca gaggatgggt ttcctttgat gggaaatctg 10440 tggcgattca ttgatgggcc tctgaattct gctgaagcag aggaagtagt aataccccat 10500 ttataatgga agtgcattct cacttaaaaa caactaatat tattctagct ggacctagcc 10560 tctagaaaca gccaaattac atttgacttg agtggattca taataattaa aaaatttctg 10620 gggcatggga taaatgtgtt aggtattgct aagtcaaggc agccctatcc cctcagcaga 10680 agtgagggaa tatgaaagtg tgtgaatgct aacataattt tggggaatat cgccgtcaga 10740 tttccagatg atattccaac atgtttgtga aacttcagtg tcttcctgtg ttcatacagt 10800 gttccagtgg aaaaataatg cttagttctg gaaggtttca gatgtgaaca ctgaactcat 10860 cgttttcttt tttgggtagt agagttagag attccatcct cttgaaagca cagttgcccc 10920 gggaagagta aaagggagca gaaggcgtaa gccaggcacg gctgttttca ctgttgttca 10980 ccttttgtat ccttacgaat atgaagatgt actaagttgt gtgttttgcg tgcatatata 11040 attttaagct acttgagttg taggtccctc cagtctgtga ttcagtttga gatgggactg 11100 tatgggaatt aacagtgcct tgtcttctta agcagtgatt tgtgtatgtg ctgatatagc 11160 tcagtatgtc tttgaaacca gttgtctggg gctaggcctg caatcagctt ttggctaaga 11220 ggtcccagga tggaacaagt agtgtgaaag aggactgata ccttggcctc acacacagta 11280 ctgctcttag actggggcaa gtgaaactcc tcacttcaga gtgccccatt ctaggccccc 11340 tcactcccaa aggggtgagg gatcactggg gccatgggaa tgtgcttgtt cagctctcgt 11400 gggctctcct tctgtaccac gttctggaca tctggagttc cttgccccaa atccctgagc 11460 ccacgtctgc gtccgcacag tctatttcct aaggtcagtc catctcctcc aggtgggaac 11520 gtgccaccat tgactgtgcc cttgggcctg agtgatggcc aagggctgtg ttggggagtg 11580 ttgtggatgg atcctggcac cgagggctgg gatatcctct caaatgaatg tgaggtgcct 11640 cccagtgctg gagagagcgg gattcaggaa gcagtggaag ggaagagcct gggatatggg 11700 gatcagctgt ctgtgccctg ctgcattctg gaataaaact ctgagggact aagaattcta 11760 aattcaaacc tgaatcaacc aggttgttac aaagataagt ttgtcagtgc aggaggatac 11820 aatatatttt acttaagtta ctagctcgat tgatcatttt taaattttta gctacatata 11880 gtatgtgggc ctccatttgt cctcttatcc caggccttgc agaatttagg aataagcctc 11940 aatacagtgt tctaacccag tgacttccgc ctcgatgtac agtagattga acctgatcct 12000 ttatacttta gtgatcatta gttgatacca gttcaagtca ggctttctag aaatctcatt 12060 gtatgttagg ggttcgatta gagtacagtc atgcatcact taatgaatgg ccacaggata 12120 cattctgaga aacgcattga tagatgattt catcattctg tgaacatcat agagtgtact 12180 tacacatacc aagatggcat agctactaca gacgtaggct ctgtggtaca ggccattgct 12240 ccaaggctgc acatctctac aggatggtac tgtactgaat actgtaggca attggagcac 12300 agtggtaagt atttgtgtat ttaaacatag aaaaggtata gtaaaaacag ggtgttacag 12360 tcttaagggc ccaccattgt atttccagtc tccgttgact gaaacatcat tatacagtac 12420 atgagcacgt atctttctca cctggtacta gtggaaagct agaaggctta gaagtctacc 12480 tgtaaacata gcttaagtaa taatacagcc ttatttttaa atgataatag caataatagt 12540 gttcacttat tgagcatttt actatgagtt acttactaaa tatatttcat cgttaattta 12600 ctctttgtgt tatttgatct ataacatcgt ttaacaggga aattacctag tacataatgt 12660 actgttatct acattttatc tagatgagga aactgaggca cagagaaatt aagtactttg 12720 cctaggatta cccgtgaagt taagtgacag aatcaatgaa tctggaaggt ctggcttcag 12780 atctcttgtg ctgagtcact cgcatacttt actacctcta aggtttctaa tcagaggaat 12840 ttgtatctgt attccctgct actcttaccc tctatgtggg atttggcctt tctccattat 12900 ccctgtgaac tcgctctggg accttccttc ttgtacttgg aaccatcaga aagtgatctg 12960 agaacataga aatctactgt gttgtgaaac agaattacct ggaagcggaa aaagccctcc 13020 tggctcaatt cacatgtcac ggcttatggt cgtatccggg gaacatatga aactgggcac 13080 tgagtgcgga gtcaggaaag ccctgtccat cctctgggtt tctggggaaa acgtggaccc 13140 cttcattgtc actttctcct gtatattttt gtttttactt ttagaactgt acaattacgt 13200 aataaataat aaaaagtcgt tggaaggata ggtgaagttc agaagtgaaa gtgttttgga 13260 ggagtctaag ctccttccca ccctcattga cctttcctct ctaataaata gaactggtct 13320 aaccaaggat ctgtggaatg agcagagtcc aacggagatt cagggattct aataacctct 13380 tgtagaatca ctggtttgtt tcagccacaa gaaggaatta ccttttgaca ttggcttgaa 13440 cagctgttgt gcaaagaaaa actttttgga aagttctgga agtaccagat tgattttata 13500 ggtttttttt tttttttttg gagggacatg ggggtattga cagttgatgt taatcagaaa 13560 tcctaaatta tgtgtattcc tggtatgttg caatcagccg gccacctggt tttcctctgg 13620 gctcttaatt ttaggtgtat tccgaggaag tttttctaac ttttctgtaa acacagacca 13680 ggtatattgc atactttcaa tgtttaacca aatctcttca ctgtttgcag tattatctgt 13740 aggctctcat gttttaagac ttccccatgg tgtttttgta ttgtattttg ctaacctata 13800 aacaattctt tgaacttaaa acaagatatt tgggcagtaa caataaattt taaaaacatc 13860 aattcaactt ttttacatta gggcttggac tatggaaaaa gtattgggca gcatgcctca 13920 tactgagttg tttaatgaat ttaaaagtat agccnnnnnn nnnnnnnnnn nnnnnnnnnn 13980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14520 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14640 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14700 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17520 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnggt ggagagttct gtagatgtct 17580 gttaggtctg cttggtccag agctgagttc aagtcctgga tatccttgtt aaccttttgt 17640 cttgttgatc tatctaatat tgacagtggg atgttagact cgcacacaat aataatgaga 17700 gactttaagt ctttttctag gtctctaagg acttgcttta tgaatctggg tgctcctgta 17760 ttgggtacat atatgtttaa gatagttagc tcttcttgtt gaattgatcc ctttaccatt 17820 atgtagtggc cttctttgtc tcttttgatc ttagttggtt taaagtctgt tttattagag 17880 actaggattg cattccctgc tttttttttt cgcttggtag atcttcctcc agctgtttat 17940 tttgagccta tgtgcatctc tgcacgtgag acgggtctcc tgaatacagc acagtgacgg 18000 gccttgactg tttatccaat ttgccagtct gcgtctttta actggggcat ttagcccact 18060 tatatttaag gttaatattg ttatgtttga atttgatctg tcattatgat gtttgctggt 18120 tattttgccc attaattgat gcagtttctt cctagcctcg atggtcttta caatttggca 18180 tgtttttgca gtggctggta ccagttgttc ctttccattt ttactgcttc cttcaggagc 18240 tcttttaggg caggcctggt ggtgacaaaa tctctgagca tttgcttgtc tgtgaaggat 18300 tttatttctc cttcacttgt gaaacttagt ttggctggtt atgagattct gggttgaaaa 18360 ttctttaaga atgctgaata ttggccccca ctctcttctg gcttgtaggg tttctgctga 18420 gagatctgct gttagtctga tgggcttccc tttgtgggta acccgacctt tctctctggc 18480 agcccttaac attttttcct tcatttcaac gttggtgaat ctgacaatta cgtatcttgg 18540 gattgcgctt ctcgaggaat gtctttgtgg tgttctctgt atttcctgaa tttgaatgtt 18600 gacctgcctt gctaggttgg ggaagttctc ctggataata tactgaagag tgttttgtaa 18660 cttggttcca ttctgtctat cactttcagg tacaacaatc atagcattgg tcttttcaca 18720 tagtcgcata tttattgaag cctttgttca tttcttttca ttcttttttc tctaatcttg 18780 tcttcttgct ttatttcatt aatttgatct tcgatcactg atatcctttc ttctgcttga 18840 tcgaatcggc tattgaagct tgtttatgct ttgtgaaatt cttgtacttt ggttttcagc 18900 tccatcaggt catttaagct cttctctaca ctggttattc tagttagcca tttgtccaac 18960 cttttctcaa ggttttaagt ttccttgcga tgggtcagaa cgtgctgctt tagcttggag 19020 aagtttgtta ttaccaacct tctgaagcct acttctgtca actcgttaaa ctcattgtcc 19080 atccagtttt gttcctttgc tggtgaggag ttacgttcct ttggaggaga agaggcgttc 19140 tgtttttgga attttcagcc tttctgctgt ggtttctccc catctttgtg gttttatcta 19200 cctttggtct ttgattttgg tgacgtacag atgggttttg gtgtgggtgt cctttttgtt 19260 gatattgatc ctattccttt gtttgttagt tttccttcta acagaggccc gtcagctgca 19320 ggtctgttgg agttgctgga ggtccactct agaccctgtt tacctgggta tcaccagtgg 19380 aggctgcaga acagcaaata tcgcggcctg atccttcctc tggaagcttc gtccaagaag 19440 gacacccacc tatatgaggt gtctgtcggc ccctactggg aggtgtctcc tcccagtcag 19500 gctacatggg gctcagggac ccacttgagg aggcagtctg tccgttactg gagttcaaat 19560 gccgagctgg gagaaccact gctctcttca gagctgtcag gcagggatgt ttaaatctgc 19620 agaagccgtc tgctgccttt tgtttagata tgccctgccc ccagagatgc aatctagaga 19680 ggcagtaggc cttgcggtgg gctccaccca gttcaagctt ccttgctgct ttgtttacac 19740 tgtgagcata gaagtgcgta ctgaagcctc agcaatggcg gggaggcgct tcccctcacc 19800 aagctccagc atcccagctt gatctcagac tgcttggcta gcagcaagca aggttccatg 19860 ggcatgggac cccccgagcc aggcactgga ggcaatcacc tgctctgcca gttgcgaaga 19920 ctgggaaaag cacagtattt gggcagagta tactgttcct ccaggtacag tcactcacgc 19980 ctttccttgg ctaggaaagg gaaatcccct gaccccttgc acttcctgga tgaggtgacg 20040 tcctgccctg ctttggctca ccctccatgg gctgcaccca ctgtccaacc agtgccaatg 20100 agatgaacca ggtacctcag ttggaaatgc agaaatcacc catcttctgc atcgatcttg 20160 ctgggagctg tagaccagag ctgttcctac tggggcatct tggaagcaac tctgggtctg 20220 agtttctgtt tgttgccctg atgtatatcc ccagtgccta gaatgatact tgttacatag 20280 gaagtgcttg atccatgttt gcacaaatga atctttctca taatgaggtt tctctaaaca 20340 agctgttctc ccaaaaactt acacccagct ttatgttgaa gcatctcatt atacattgga 20400 aagatgaaat gtgtagtgag actttgaatc ttcttttgaa tctagaaaca ttagcatttt 20460 tagaccattc tattttaata tttatgaaat ttatgaaata ataagaaaca tgaggccggg 20520 ctcagtggct tatgcctgta atcccagcag tttgggaggc cagggctagt ggatcatgag 20580 gtcaggaatt tgagaccagc ttggccaaca tggtgaaacc ccacttctac taaaaatata 20640 aaaattagct gggcgtggtg gtgcatgcct gtaatgccag ctcctggaga ggctgaggca 20700 ggagaatcat ttgaacctgg gaggcggagt ttgcagtgag ctgagatcgt gccattgcac 20760 tccagcctgg gcaacattgc gagactccat ctcaaaaaca aaaacaaaaa caaaaaaaat 20820 gtgtgaccta aattaggctt atagatgaac cattgcagtc atgattaatt ccgccattgt 20880 ttgccttgtg atctttggtg ccatgtctgt acatatttca tgatttctgt gtttttacgg 20940 tttccatttc agatctccct tgagtttaga aatctggctg agaaatacca aacagtgatt 21000 gccgacattt gccggagaat gggcattggg atggcagagt ttttggataa gcatgtgacc 21060 tctgaacagg agtgggacaa ggttagtctc ataaaacagt gtctgtgtgt gatgtattag 21120 acagagctgg cagtcctcat agtgaagctc agaacaagaa aagttgtcca gtattttcag 21180 cccctctggt tttacaattc atctgtttag gttgaatgtc tcatcataaa cagtttattc 21240 cagagttaat tccaaaccag cagctatgta ggatatcagc caggctagga gtagggtact 21300 ggagagaagt gcttatctag acaaagggat gtaattgacc atgaagatta aaactacaca 21360 tcaaaacata aggtagggtt aggagtcttg cctatttttc ataggaatgg tgtttgtgag 21420 acttactcat cacttctgtg gaagtaaaga cattttattt atttatttta aagccagtca 21480 gatttagcag gcagagacat ttcagacatc taaagtgttg atgtatttca tacctttaac 21540 tgtgcttaaa ttaggatctc cgaaaagatg ctgctacatg gtcactacgt tagtgtaggt 21600 ccaaggtctt gggcctctta atttttcaaa cctcaaaact tgacagcagt tatctttgga 21660 actgctgatt tgtgcttcct aagttaacag catacaatga ctgctagaaa tcaatttctg 21720 catttaaggt gaagttagcc gggtactatg gtttacctgt aatctcagca ctttgggagg 21780 ctgaggtggg aggatcattt gagcccagga gttagacaca agcctaagca acatagcgag 21840 accccgtctt tcaaaaaatt aaaaaatgag cagggaattg gtggcatgtg cctgtggtcc 21900 ccagctactc tggaggctga ggtgtgggag gattgcttga gcccaagagt tgaaggttgc 21960 agtgagccat gattgtgcca ctgcactcca acgtgggtga cagagcaaga cacctactga 22020 aagaaaataa agttgaagtt aaaacttctg gccaagaacc agcactggtt atgatagtaa 22080 ctcattttct gttgtgcaga tttattcagg aaacttaatt ttaggttgtt gaatagaagt 22140 tttgatcaga taaaattgaa ttaaaaaaaa ttttttttga gacagggtct tgctgttatc 22200 caggctggtg tgtagtggtg tgatcacggc tccccgcagc ctcaacctcc tgggctcagg 22260 tgatcctccc acctcagcct accgagtagc tgtaactaca gtgcatgaca ccataccagg 22320 ctcatttttg tacatttttt gtagagagag ggttttgcca tgttgcccag gctagtctca 22380 aactcctggc atcaaacagt cctcccactc tggcctctca aatgttggga ttacaggcat 22440 gaccagccaa ttatttcaag gagttatttt ttttcttcta ctttggggga agatgaatta 22500 tataagtctc cattttagga gtatttctac caaaagaact attatcttca aatatatttt 22560 tggatagtac tatagatata ctaatttttt tttaaatttc tagtaattct tttgaagatt 22620 ttgtatagct gtccaaagcc aatttctgtc tacctaattt cagcaagatt tcactctttt 22680 catgttactt ttgtcccaga acaaatttca agtgctttct cttcacctgt gcattcttcc 22740 ccctgattag tctctggctt tgtattactt tcagtcagag acgacttttt ttttttgaga 22800 cagggtctca ctctgtcacc cagactggaa tgcagtggca cagacaaggc agccttgacc 22860 ttctgggctc aagcaatctt ccttgccctc agcctcctga gtaactggga ccacaggcac 22920 gttgccacca tgcctggcta atttatttta atttttatta tttttgagac agggtattgc 22980 tctgtcaccc aggctggagt gtagtggcat gatcaaggct cactgcagcc ttcacctcct 23040 gtgctcaagc agtcctctca cctcagcctc cccattagct gggactatag gtccacacca 23100 ctacaccagg ctaatttttg taattttttg gtagagacag ggtttcatcg tgttgcctag 23160 gctggtcttg agctcctggg ctcaagcgat tcacctgcct tagcctccca ggtgtgagcc 23220 actacactca gccttttaaa attttttaca gagatgaggt cttgctttgt tggccaggct 23280 ggtctaaaac tcttgggctc aagcagtccc ctctccacag cctcccaaaa ttccgggatt 23340 acaggcgtga acttcggtca tttcctaact tttacccttc ctaatgacac tccagagctt 23400 accttcttta cttttgcttc ttaagttaac taatagacaa ttattgtatg tggatattgc 23460 attaagttgt cttaggatac ccttttcaga ggaggacagc ttttgacaaa ttgctgtcgc 23520 ggaaaaaaaa agtatttggc aattaagagt tgcatttact gaaatctctg ttgagagagg 23580 ggaagttacg ttgtctctaa aagaaaaact aaaaagaaaa ggggaagttt tagcaaagtt 23640 gttaaagcct gacacttaag tcatactacc tagttttgaa ctcttagccc ctgccacaga 23700 cacggcagcc ccttgaacct tcctgggttc aagcgagcct cctacttcag ccccctgagt 23760 aactgggacc actggcctgt gtcactgtgc ctggctaatt tttttttttt cctcacatgg 23820 gcaatgttgg gcaagttaaa tcgacttctt tgtgcctcag tttcctcatc tgaaatggag 23880 atcatactgc tatgtacctg atacaatgtt tgtgaggatt gaatgtgcag agttcttttt 23940 ttctgttgtt gttgttttga gacggagtct cactctgnnn nnnnnnnnnn nnnnnnnnnn 24000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnna tctcgtgatc cgcccgtctc agcttcccaa 24120 agtgctggga ttacaggcat gagccatcgt gcccggctga atgtgcagag ttcttaaaac 24180 cgtgtcaaga acataaaata gttatttgtt ctttcatata atgatgattt tgagggcctg 24240 cggatcttga catgttatca gattggtcaa aaaaagatta aaccatagtt ggtattgtcc 24300 tagttcctgt taccagaata ttccatcttt catcgttgcc ttctctcata gttttatgta 24360 tcaaaaagtt tattgtaaag ctaggccggg cacggtgtct tgggctggta atcccagcac 24420 tttgggaggc caaggctggc agatcagttg aggtcaggag ttcgagacca gcgtggccaa 24480 catggtgaaa ccccgtctct actaaaaata aaaaattagc tggatgtggt ggtgggtgct 24540 ttaattccag ctactcagga agctgaggca ggagaatcac ttgaacccaa gaggcagagg 24600 ttgcagtgag ttgagattgt gccactgcac tccagcccag gggacaaagt gagacttgat 24660 ctcaaaaaaa aaaaaaaaaa aaagttattg taaagctaga cacggtggta tttgcctaca 24720 atcccagctg ttcgggaagc tgaggcagaa agattgcttg ggtccagtag tttgagtcta 24780 acgtgggcaa atatatgaga ctccatctca aaaaaaaaaa taaaaaataa aaataaaaaa 24840 atgtttacta gtttttttca gtagcctttt attatagtag cagtacatgt gtattgtaga 24900 aatttggaaa atacaagtga aaaataaaaa catcaaattc ccgtcagcca gagactgctg 24960 tgaaatgttt tgagcacatc cttcttgaat gttttttaaa tcctggtatg tatatttgta 25020 ttttaaaatc aaaatgcatt cttacccatt ctcttttgaa cctgcttttt tgtagctaat 25080 gatctctagt gtgtccattt cagtaaaaat tccattatta aagtgcttta aaaatcgtct 25140 cttacagtac tgccactatg ttgctgggct ggtcggaatt ggcctttccc gtcttttctc 25200 agcctcagag tttgaagacc ccttagttgg tgaagataca gaacgtgcca actctatggg 25260 cctgtttctg cagaaaacaa acatcatccg tgactatctg gaagaccagc aaggaggaag 25320 agagttctgg cctcaagagg taacagattc agggtatttt gggggaaaat aactttagac 25380 attctctgaa aaatccttta actcttgtgg ttgcgggtga cagaaaaaca agccaggcct 25440 cccccaggca gcataagggg atgtggaaaa taggatagat tgacatgagt ttgcttcagg 25500 tagactggct gactcccagg attcacacca cgtaatcagt atattcaagc cttgctgtcc 25560 ttgatttctt tcagacggtc tttctccaag tggtggatat ggtaacaacc cacgtgcact 25620 agcttaacaa aaagttctta ggaatggctt tgttcggcct ggcgcagtgg ctcatgcctg 25680 taatcccaac agtttgagag gccaaggtgg gcggatcacc tgaggccagg agttcgagac 25740 cagcctggcc aacatagtga aaccccgtgt ttactaaaaa atacaaaaat tagccgggcg 25800 tggtggcaag ggcttgtaat cccagctacc tgggaggctg aggcaggaga atcgcttgaa 25860 cccaggaagc agagattgcg gtgagctcag attgtgccac tgcactccag cctgggcgac 25920 agagtgagac tccctctcaa aagaagagga agggcttggt tcttctgctc agccctgaat 25980 cagttactgt tgctacacag ctgagttctc tggcctcacc tggattacgt ctacacagta 26040 cacacagaat ggatttcccc caaagaaaga attctgcggc aggaagggga aagggatggc 26100 aggtagacaa aaactccagg tgtctgtaat aagggacagg gtcgatcttt aattaaaaca 26160 tggacaggga acagaaagct tttgatactg attttgttca gaaggaaagt agaaaatttt 26220 atgactgttc cctgaattta ttccagcatt taccttttgc tttccataaa agtgtttcct 26280 gcagccaagt actttaaagt tttaaaaaga cgggtgaggc taagtgtggt gtctcatact 26340 tataatccca gtgctgaggc caggagttca agaccagcct gagcaacaca gcaagatacc 26400 atctctataa aaaattgtta gaaaatgatt ctgctgaaag agcaaaaata aaaattaaag 26460 aaagtagaaa aaataaaact aaatttaaaa gattaactgg gcatgttggc atgcacctgt 26520 attcctaggt attcgggagg ctaaggcaca aggatccctt gagcgcagga gctcaaggtt 26580 ggattgagtt gtaatcacac cactgcactc cagcctcggt ggcacaatga aactgtctca 26640 agaaaaaaaa aaagtgacag agggaaacaa tatttgcaat tcatagagca gatacagggt 26700 tcatattcct aatattaaaa aaaacttcta aaagttaaga aaaaggccaa ctgccccaca 26760 gaaaaatggg caaggagata agaacaagat tgttcacagg aagagacaca cagatgatta 26820 ttaaaaatct gaaaagatgc tgagtcttac tcctaagaaa aattcacatt taaactactc 26880 tgggggctgg gcaaggtggc tcacgcctgt aatctcaaca ctgggagacc aaggcaggaa 26940 gatcactgaa gccagggtat cgagaccagc ctggacaacg tagtgagacc ttatctctta 27000 aaacaaaaca aaacaaaaca aaacaaaaaa aacagtaaaa attggccggg cacagtgact 27060 cctgcctata atcccagcac tttgggaagc ccaggtgagt ggatcacttg aggtcaggtg 27120 tttgagaaca gcctggccaa catggcaaaa ttccgtctct actaaaatta caaaaattag 27180 ccaagtgtgg tggcatacgc tggtagggcc agctacttgg gaggctgatg tgagactcca 27240 tttaaaaaaa aaaaatcaaa aattagctgg gtatagtggc acacccctat agttctcgct 27300 ccttgggagg ttgaggcagg aggattgcct gagcccagga gttcaaggct gcagtgaacc 27360 atgatcacac cactgcattc tagcagcctg ggagacagag caaaaccctt gtctcaaaac 27420 aaacaaacaa caacaaaaac aaaaaacact tccctcagct cagacatggc cttttaagtt 27480 tcctaggtga ctcgtgtgca gccagggttg agaaaccact cttgtcttac ccctcttttg 27540 cagacacagg gctcagagaa gggaagggga ttgtctgggg atgtatagtg aggcagtggc 27600 tgccttggaa gtggagtctc agtctcccgg ctcctaggcc agcccctgac cactgttcca 27660 ttgtctccca gacagaacat cagccacggg catgtgatgc atgagcgtga gccacaccat 27720 cttgcacaca caggagcaga gccctgctct tctcattcac ttactttatc tgtaaaatag 27780 catcatttct accacacggt ggtggtgtga ataaaatgag atgaacttct agcatagagt 27840 gcttagtaaa ggttctggac atttcgtagt agttgaatca tgccaaatgt ggtcctaggt 27900 gattggcttc ttttgctagc atgttttcag ggctcctcca tgctggggca ttgcatcact 27960 gctttattcc tttttatcgc ctagtattat tccactgtgt ggatagacca catttatcca 28020 ttcatcagtt ggaggatatt tgggttcttc ccattttttt tggctatggt gaatagtact 28080 gtgtacattt gcatataagg ttttgtgtag atgtgtgttt tcctttttct tgggtctatg 28140 ctgagaagtg gaattgctgg ttcatacagc agctcgaacc ttgtgaggag ctgccagacg 28200 cttttccaag gtcgctccac cattttacat tcccgtcagc agtgtgagag tcccagtttc 28260 accagcactt gttgttatct ctttttaact gtatgtatat atacttaaca ttttatttat 28320 aataaatgta cataatagag aatttgccat tttaactatt tttaagtcta ttattcagtg 28380 gcattaagta cattaatgat gttatataac catcaacact atgtttccag aactttcgct 28440 agcttcagag aatcctctaa ataatatcat taaaaatcat caagccgaat cccactgtta 28500 gaattaaagg ttttatttca ctttcaagtt atcaggatcc agggaggtgt aatacactta 28560 gaggatagac tcagctcatt tcccagctat gcctttcagc agcattctta ccagagtagg 28620 aatataatgt tagtcattat ttagaggcct ggccatcttg agaaggttta ctgtttagtc 28680 tgcagtacaa ttataactgt ttttgtatat tgggttattt ttttcagaag taggccagta 28740 gctctaacag gagcctcttt agcctgaatt cgtccaagta gtgcagtgtt gcactagttg 28800 tccctcggga catgctcccc aatacgtaac tcacttccag gttgcaactg gacacttact 28860 ggtagtcaga aatagctatt gcatggagct taaaatgaac ttgatcttcg tgaaagatga 28920 gtctgcagct aagagacttt actgtatatc atagtgtttt tttttgtttt gttttgtttt 28980 tgtttttgtg acggagtctc actctttcac ccaggctgga gtgcaatggc gagatcttga 29040 ctcactgcaa cctccgcccc ctaggttcaa gcaattcttc tgtctcaccc tcctgagtag 29100 ctgggattac aggcgcctgc caccgtaccc ggctagtttt tgtattttta gtagacacag 29160 ggtttcacca ccttggccag gctggtcttg aactcctgac ctcgtgatcc accctcctcg 29220 gcctcccaaa gtgctgggat tacaggcgtg agccacggcg cccagcctgt atcatagttc 29280 ttatgcacaa agacccttta atattgtttg taaattctcc cctatgcaca cgctgacctg 29340 ttccttaatc ttcttatctg tctaggtttg gagcaggtat gttaagaagt taggggattt 29400 tgctaagccg gagaatattg acttggccgt gcagtgcctg aatgaactta taaccaatgc 29460 actgcaccac atcccagatg tcatcaccta cctttcgaga ctcagaaacc agagtgtgtt 29520 taacttctgt gctattccac aggtagggaa cggggctcct ctgggtggat acggggctaa 29580 agggagtggg gtaggagtaa gggtggattt tgctgtgcta tattcaagga tatgattcct 29640 taaaaagacg atgactccag tttattacgc tgggagtttc atagcacccg cctttgcttc 29700 cagccaccaa actcagctca gccttgaggt taagcctgct ccttttcaga accttctttc 29760 cggatttact attttctaca gctatcctaa actagttagg ttcttttcct cacagttaag 29820 tcaaggtctt tggcttagat ttatggggag tgctgggtaa aacctgggtg aagctgttat 29880 cattaaaaag tcttcattaa gcacctaatt actgctgtcc ttttcctaga cccggcataa 29940 aaagaacctg gtccggtaga cctagcctct cagtatgcta ggaacttaca ctttttagtt 30000 gcctttacca agtattgcag atactactgc aaataagtga agaaagtaac agcatttaac 30060 tgatttggga acttggtttg atcttgttct aatgacccac ttcgaatggt ggttgaaagt 30120 aaaatctgta tcgccgtctt atgtttccat ttacctagaa atactttacc tttgagcaca 30180 ggaaattaat ccccttctgg ttgttctccc cctggcattg gttttaaata tataatgatt 30240 atgtttgttg taggaaaaat agaaaaacaa ctacaataga aaattcttcc catatattat 30300 tttgaaatac atatttccga tccgataatc cattgctcta gcatggaaaa tgttggattt 30360 acttgtgttt gctttttcca aataaaatgg aacttttgtg gctacattat agaattgttt 30420 tagactgctt aattctgtgt gttgttgaga aagggaggag tggggaaggt aaaaatcttg 30480 acatactttc ttcgtgggta ttttttcttg agcgattcca tcttagttga ttagcagtta 30540 gcaattgccc attcaacaga aggttttctt acctttttgt gataatgata gctaacgaca 30600 tcatttcttc ttttttccct ctcttcttgt tgtctctagg tgatggccat tgccactttg 30660 gctgcctgtt ataataacca gcaggtgttc aaaggggcag tgaagattcg gaaagggcaa 30720 gcagtgaccc tgatgatgga tgccaccaat atgccagctg tcaaagccat catatatcag 30780 tatatggaag aggtgggttt ttatttaact acttggataa tttgtagcta cttttatgat 30840 ttagtaatgt cactgtttaa ccaggtttgg atattagatg atcctaacaa ttcactatcc 30900 tgtggcctaa agagacagga attgatatcc tttataagga aaaaagtcta ttcacaggag 30960 ccgagcagat tgctcactgc tgtgtagtac cctggtgaga ggagataaat ggagcaaggc 31020 tgtaggttgg agcccctcag tagaatcata gattttgagc tgcaagatga tgcaggaggc 31080 caaccaagct tcttgttgct ggtgaggaat gtgaggttga agcttgtctg tgctgatgca 31140 gtgcgtgatt gagtggatct ctggctcccg tccatgtgtc ctgacaccca gtctggtact 31200 ttcattatgc cacaggcctc aattgaaaaa tcacagtagg gaatttaggc caaggaaagc 31260 catcaagttg caattatttc ctaaattttc tttggaaaat ttcatttcaa ataccaaaac 31320 catcctataa aaagaaaact taccttctta ggtcaaatct ctaatatttg actaggttca 31380 aaaagtttat ttctggccag gcacagtagc ttactcctga aatcccagca ctttgggaga 31440 ccaaggtggg aggatcactt gaggccagga attcaagacc agcccgggcg acatagcaag 31500 accccatttc tacaaaaaat ttaaaaattg tcatggtggt gcacgcctgt ggtcccagct 31560 actcaggagg ctgaggcagg tggatcacat gagcctgaga ggtcgaggct acagtaagct 31620 gtgtgatttc atcattgcac tctagcctgg gtgatagagt gagactttgt ctcaaaaaaa 31680 aaaaaaaaaa aaaaagtctt agagaccaga agtctctgta atctctaata atctctaggc 31740 cctagagcag tggtttgtaa atggaggtga tttgctcccc tccccccaga ggacattgga 31800 caatgtctgg agacattttt gattgtccta accggcagga atcgggtgct actggcatct 31860 ggtgagtaga ggcccaggat gatgctgtga tcctcaggtg tgatcctgtt gagaatgaaa 31920 cactgtagac tttatgaaaa catacaagac cctcatcatt tttcctttgc ctgagctccc 31980 tccccagagg ttacctctgt tcatggtttt gtgcatccgt ctagtccccc tgttacgcgt 32040 ttacaggaat atggtttgca acagtgtttt catctaaata gaattataca aaatagcgat 32100 ttctgatttc tcttgcatat tgcacattct tcttatactt cctccctacc tttatctgac 32160 acagaaatgc tgtatgtcca gaacttctat cagaggcacc tatggaagtc taagggaaga 32220 ccacatcgct tttaaaaacc ctaaaatttt gtagtcacta gatgaaaata ttcagccagt 32280 gacccaaaaa attgctacca atgagactct ccattttgcc atgtagccag aacttacttt 32340 gatctatgtg cctggggtag tgaccaagta ggtgggtagg agtaatctca gggaaacttg 32400 aggccccagc ctcatggcta gggtcataat ttgaacccag gtctgtctga catcagaatc 32460 catgatgtta accccaattc taaggggttc aactaccctt tctaaatgga atcctgctat 32520 attaagcact atttattcat tttatataaa ctagaaacat tttatgtagt aagtagttga 32580 gagtgttttg gttttgcagt ttgatcacta gttttagaaa ccagttttta aacactttgt 32640 ggccaattcc attactatat taaaattcag atttatttgg tttttcctta actattggga 32700 ttaaatcctg gttgtaattc atagtttgag ggcgagggtg ggcagtctac atttggctga 32760 gccctgtttt tgtgaataaa tgttatcaga acacagccac acccatttgc ttctatgtct 32820 tctgtggctg cttttgcaat gtgacggccg agttgaggag ctgcaacagg cgatgacttg 32880 taaagctgaa aatatttttt ggcccttgaa taagaggttg gctgacttct gacttagggc 32940 atcagttgtt ctgttatccc agtaaaactc aaggcattag gggagaaatg ttaatattaa 33000 tacttaagtt gatttgattt agggaaatct ttgaagattt ctaagtctta agcagtagaa 33060 cctgttaatg gttttagttt cagcagtaag gacattttac aagtaaagtt ttaaatgaaa 33120 acattttgta tgaagccaca agtcgtctgg cctcttgctg gtgtccagat attaacactg 33180 atcctatttc tccttgctga ccaagtctgt cctttgtagt aagaaaggaa gaaacgttga 33240 ctctgtccga tctctggact tagtgttgta gcgagcatgc acctggaagg gacttgccag 33300 aggacctcct catgcttctc cagtgcttag tgggggcttg gagtgcagcc ccaggtcttc 33360 acgagcagtt ggccacactg cagggccctc accccactct ggagcagcct ctgcttcaaa 33420 ccagcctgga tgcttgtcag ctggggagaa gatcaacctg ctattttggg atagaaataa 33480 atgctcagcc aaacggccag aaacccccat tcccctctct gccaaagtga attccttggc 33540 agggagaagc ttgttcgtgt ctctgcacac ttcctgtgcc ctcctgtggt taagtcagag 33600 aatcatccgg ctctttgagc cccaggtgcc tagctgctca aggatggtcc ccagccagca 33660 gctgccagga atcacctggg agcccattaa gacatccagc ccccacccaa acctatcgaa 33720 tcagaatctg cctttttttc ccaaatgatg tttttgcttt aatggaagtt tagatgttca 33780 tagacaagag ttttaaatga tgatcaagct gattccatat tcgcagttgt aagtagaact 33840 gctgagacgt ggaagtacca catggactca cagaggagct gctgtatgta gcacagcatt 33900 gcacaagagc ttatttcagt ctagtaaaca tttataggag cctgtgtcat ttaatcatca 33960 agcctcgcac tgtggctcac acctgtaatc ccaaaacttt gggaggctga ggcaggcaga 34020 tcacttgagg taaggagttc gagaccagcc tggccaatat ggcaaaaccc tgtctctact 34080 aaaaatacaa catttagcca ggtgtggtgg tgcacacttg tcatcccagc tattccggag 34140 cctgagacat gagcatcgct tgaactcggg aggtggaggt tgtagtgagc tgagatggca 34200 ccactgcact ccagcctggg caacagggtg aaggcccttt ctcaaactcc tcaagtattt 34260 ggcttcaact ttatgccggg catgtagatg aaaagtcggc tatgacctgt ccttgacaag 34320 cagatgtaac tccttgattg aggctagtag gtttttaaga cctgaataat tgagtttgca 34380 gaaacctact gtgtgccttc aggtaaatgg agagtggggt ttggtctagc aacgaagcat 34440 ctagaaggtc tctttggcct taccggctct gttttaggta agtccacgtc tgagtaccag 34500 tgactgcagc tcttccagtt gtgctgtcat gtttatatgt tagaaatgat catcaaagga 34560 ctcaaaagtt ttgccactaa ttgtattacc ggggactgtc acaaccaaga tttctcttaa 34620 tttattcacc ttacttatct cctggaaggg catattgaag tgctcttgga gttctctaaa 34680 agggtttttg ttggttgtgt atattcactt gggtgccagc gattgattcc aaataagtaa 34740 atcttttttc ccaaaaggat gtaagatggc ttatggttat aagtacaaca ggctaacaaa 34800 gtacaagtag atgagaaagt aaaatgaaga aataaagtca taggagccac agaattaacc 34860 caggaatgaa taagtgtgta gtttggtgct gatgttatca tcctttattt gtacattgct 34920 tgtacagttg ctctgagaag gtaagtctta aattttcaaa agtgaaatgt caccgagcat 34980 ggtggctgat gcctctaatc tcagcacttt gggaggctga ggcaggcgga tcacttgagg 35040 tcaggagttc gaaaccagcc tgacttatgt gatgaaaccc tgtctctact aaaaaaaaaa 35100 aaaaaaaaaa aaaaaaaaaa aaaaatccaa aagttagttg ggcatggtgg caggtgcctg 35160 taatcccagc tacttgggag gctgaggcag gagaatcgca tgaacctggg aagtggaggc 35220 tgcagtgagc caagattgca ccactgcact ctagcctggg tgacagagcg agacaccatc 35280 ttaaaaaaaa aaaaaaatct acaatatacc aaaaccatta cttacctgag aaactattct 35340 cagggtcatt gtagtgaatg cctattttat ggcttttgat ggcatcaggg cactcaggtc 35400 atttacaaga gtagtgtgtg agaccctgtg tgtcactgcc actcatcttg gccttcggcc 35460 actgctgtag caaccagttt ccaagtaggg ctggaccttg ccttctgctc cagagacctc 35520 tcgcttcctg cccttgggct tctgacgagc tgcaggaact gcctggcacg tgggtcccca 35580 caacccagag gaggtgaggg ccacctctct gctcctcagg gccacctttc ataaggctcc 35640 ttgaaggtcc ctcaagatca agccaactca acacatcctt gataggcctt cctgccttct 35700 gtttcacttc tccactcgtt tccaaataaa tggctgcatg caagcttttg cctcaggttc 35760 tgcttttagg aggaaggcta agacaagcag taaagcaaca tgggcaggca gaaggatgac 35820 ttctaataga attatctcat cactatatat tttactttat ggatgcttgt attgaaaagt 35880 cttggctggg tggagtggct cacgcctgta atcccagccc tttgggaggc cgaggtgggt 35940 ggatcacttg aggtctggag tttgagacca gcctgaccaa cactggtaaa accttgtctc 36000 tattaaaaat gcaaaaatta gccagggatg cacgcttgct gtgtgccagc acagggctag 36060 gctggagata aaaaggtgag taagtaggtg cggtgtagtc agggtgaaaa ctacagatgg 36120 tccatttcca cgtaagtgga aaggtaaagg tatgtacaat agggtggctc ctggctgaac 36180 ctggagctgc agacaggttt tctagaaggc ataatcctga agttgagact tgggggccta 36240 ggtaggagcc agttgaaggg acgtgggagg cgcattccag agagaaggag tggtatgaga 36300 ctggaacaga ggtgtgcagc agcatcgcat gggcgaaaca acagtagaca gttgttcttt 36360 tgtttttgtt tgttttttga gacagggtct tgttctgtca tccaggctgg agtgcagtgg 36420 catgatctcg gatcactgca acctccacct cccaggctca agtgatcttc ccaccccagt 36480 ccccaagtag ctgggggacc acaggtgcat gccacgatgc ccggctaatt tttgtacatt 36540 ttgtagaaac agggttttac tgtgttgtcc aggctggtct taaacgcctg agcttaagca 36600 gtctacatgc ctcagcctcc tgaagtgctg ggattccaaa catgagccac tgtgcctggc 36660 ccggcaactg ttactagact atagagaggg aggtgggcaa gggctggtga cactagacag 36720 gtgcagtagg tctggaccat gggtggcctt gcgctacaca ttacagagct caggcttttt 36780 ttctccaggt gagagggctg gtgccactga ggcatcaagc agaggtttga gatctccttg 36840 gtgacagtgt agagcagaca ggtagatttg ggaatttaag cttagactca cgttggagac 36900 tgagatagct catctgagag gcactcaggg cctaatctca ggcagtaatt ttagggatgt 36960 aggggaagag atggattctg cacatacttg ggaggcttgt ggaggagtgg ggagggaggc 37020 acagggagga ctccagggtg gttcatacgg ctccctgctt ctgttcctgt ccccctttgt 37080 caagctgtgg tctgtactgc gtgttccatc ttgtttctaa gctgcttttg cccagtcttt 37140 ccagcatttc cctttcgtca tgttagtctg tgcctgtcta cgtgaactat ggtgacgttt 37200 attgggcctg gcactgtgag gtgctgggga tgtgaagatc attgtggctc agccgctgct 37260 ctcgagggcc tctgggtgca gtatgcacac ctgtgcctcc tgtttgctca ggaagacagg 37320 ctttgagatg agctggggct gacatcccca ccttatcatt gggatggctt tgggtaagtt 37380 atgttcatgt tctctgagcc tccctttcct cattggtaaa atgggtataa aatacctgcc 37440 agtggagggt tgttgtaagt agccatggaa aatgtaaagc acatagcact taccattttt 37500 tcctgtgtct ttaacagatt tatcatagaa tccccgactc agacccatct tctagcaaaa 37560 caaggcagat catctccacc atccggacgc agaatcttcc caactgtcag ctgatttccc 37620 gaagccacta ctcccccatc tacctgtcgt ttgtcatgct tttggctgcc ctgagctggc 37680 agtacctgac cactctctcc caggtaacag aagactatgt tcagactgga gaacactgat 37740 cccaaatttg tccatagctg aagtccacca taaagtggat ttactttttt tctttaagga 37800 tggatgttgt gttctcttta tttttttcct actactttaa tccctaaaag aacgctgtgt 37860 ggctgggacc tttaggaaag tgaaatgcag gtgagaagaa cctaaacatg aaaggaaagg 37920 gtgcctcatc ccagcaacct gtccttgtgg gtgatgatca ctgtgctgct tgtggctcat 37980 ggcagagcat tcagtgccac ggtttaggtg aagtcgctgc atatgtgact gtcatgagat 38040 cctacttagt atgatcctgg ctagaatgat aattaaaagt atttaatttg aagcaccatt 38100 tgaatgttcg tactagtaga aaatgatgtg aattttcttt ctgttcggct cctatttttc 38160 tcatcatttt gttttcttta attgggttga atggagtaga tagaaatatt tatggtttag 38220 gtaacagtta gatgtttcct aagaatgcaa actgcctttt ccacacaaag gctgggaata 38280 aaattctggg tattctcgta ttctcattta aaggagttta gctttcagag agaaacagca 38340 ggattgcttt tgacctttta gaagattggt ctccagtaaa ggtggacatt tttgagattt 38400 ttataataaa gaatttaatt gctctgcatt tgtcaagtac agttcgcttg aaagcctgcc 38460 tgactgtgga aaagatggag ctcaagaatg gagttgatgg cccagcgtgg tggctcatgc 38520 ctgtaatccc agcactttgg gaggctgagg cggtcggatc acgacattag gggatcgaga 38580 ccatcctggc taacacggtg aaacccccgt ctctactaaa aaaaaaaaaa attagccagg 38640 cgtggtggcg ggtgcctgta gttccagcta ctcgggaggc tgaggcagga gaatggctta 38700 aacccgggag gcggagcttg cagtgagctc agatcgcgcc actgcactac cagtctgggc 38760 aacagagcga gactccatct caaaaaaagg aaaaaattgt aaaaaaaaaa aaaaaaaaan 38820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 38880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 38940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40080 nnnnnnnnnn 40090 4 417 PRT Human 4 Met Glu Phe Val Lys Cys Leu Gly His Pro Glu Glu Phe Tyr Asn Leu 1 5 10 15 Val Arg Phe Arg Ile Gly Gly Lys Arg Lys Val Met Pro Lys Met Asp 20 25 30 Gln Asp Ser Leu Ser Ser Ser Leu Lys Thr Cys Tyr Lys Tyr Leu Asn 35 40 45 Gln Thr Ser Arg Ser Phe Ala Ala Val Ile Gln Ala Leu Asp Gly Glu 50 55 60 Met Arg Asn Ala Val Cys Ile Phe Tyr Leu Val Leu Arg Ala Leu Asp 65 70 75 80 Thr Leu Glu Asp Asp Met Thr Ile Ser Val Glu Lys Lys Val Pro Leu 85 90 95 Leu His Asn Phe His Ser Phe Leu Tyr Gln Pro Asp Trp Arg Phe Met 100 105 110 Glu Ser Lys Glu Lys Asp Arg Gln Val Leu Glu Asp Phe Pro Thr Ile 115 120 125 Ser Leu Glu Phe Arg Asn Leu Ala Glu Lys Tyr Gln Thr Val Ile Ala 130 135 140 Asp Ile Cys Arg Arg Met Gly Ile Gly Met Ala Glu Phe Leu Asp Lys 145 150 155 160 His Val Thr Ser Glu Gln Glu Trp Asp Lys Tyr Cys His Tyr Val Ala 165 170 175 Gly Leu Val Gly Ile Gly Leu Ser Arg Leu Phe Ser Ala Ser Glu Phe 180 185 190 Glu Asp Pro Leu Val Gly Glu Asp Thr Glu Arg Ala Asn Ser Met Gly 195 200 205 Leu Phe Leu Gln Lys Thr Asn Ile Ile Arg Asp Tyr Leu Glu Asp Gln 210 215 220 Gln Gly Gly Arg Glu Phe Trp Pro Gln Glu Val Trp Ser Arg Tyr Val 225 230 235 240 Lys Lys Leu Gly Asp Phe Ala Lys Pro Glu Asn Ile Asp Leu Ala Val 245 250 255 Gln Cys Leu Asn Glu Leu Ile Thr Asn Ala Leu His His Ile Pro Asp 260 265 270 Val Ile Thr Tyr Leu Ser Arg Leu Arg Asn Gln Ser Val Phe Asn Phe 275 280 285 Cys Ala Ile Pro Gln Val Met Ala Ile Ala Thr Leu Ala Ala Cys Tyr 290 295 300 Asn Asn Gln Gln Val Phe Lys Gly Ala Val Lys Ile Arg Lys Gly Gln 305 310 315 320 Ala Val Thr Leu Met Met Asp Ala Thr Asn Met Pro Ala Val Lys Ala 325 330 335 Ile Ile Tyr Gln Tyr Met Glu Glu Ile Tyr His Arg Ile Pro Asp Ser 340 345 350 Asp Pro Ser Ser Ser Lys Thr Arg Gln Ile Ile Ser Thr Ile Arg Thr 355 360 365 Gln Asn Leu Pro Asn Cys Gln Leu Ile Ser Arg Ser His Tyr Ser Pro 370 375 380 Ile Tyr Leu Ser Phe Val Met Leu Leu Ala Ala Leu Ser Trp Gln Tyr 385 390 395 400 Leu Thr Thr Leu Ser Gln Val Thr Glu Asp Tyr Val Gln Thr Gly Glu 405 410 415 His 5 417 PRT Human 5 Met Glu Phe Val Lys Cys Leu Gly His Pro Glu Glu Phe Tyr Asn Leu 1 5 10 15 Val Arg Phe Arg Ile Gly Gly Lys Arg Lys Val Met Pro Lys Met Asp 20 25 30 Gln Asp Ser Leu Ser Ser Ser Leu Lys Thr Cys Tyr Lys Tyr Leu Asn 35 40 45 Gln Thr Ser Arg Ser Phe Ala Ala Val Ile Gln Ala Leu Asp Gly Glu 50 55 60 Met Arg Asn Ala Val Cys Ile Phe Tyr Leu Val Leu Arg Ala Leu Asp 65 70 75 80 Thr Leu Glu Asp Asp Met Thr Ile Ser Val Glu Lys Lys Val Pro Leu 85 90 95 Leu His Asn Phe His Ser Phe Leu Tyr Gln Pro Asp Trp Arg Phe Met 100 105 110 Glu Ser Lys Glu Lys Asp Arg Gln Val Leu Glu Asp Phe Pro Thr Ile 115 120 125 Ser Leu Glu Phe Arg Asn Leu Ala Glu Lys Tyr Gln Thr Val Ile Ala 130 135 140 Asp Ile Cys Arg Arg Met Gly Ile Gly Met Ala Glu Phe Leu Asp Lys 145 150 155 160 His Val Thr Ser Glu Gln Glu Trp Asp Lys Tyr Cys His Tyr Val Ala 165 170 175 Gly Leu Val Gly Ile Gly Leu Ser Arg Leu Phe Ser Ala Ser Glu Phe 180 185 190 Glu Asp Pro Leu Val Gly Glu Asp Thr Glu Arg Ala Asn Ser Met Gly 195 200 205 Leu Phe Leu Gln Lys Thr Asn Ile Ile Arg Asp Tyr Leu Glu Asp Gln 210 215 220 Gln Gly Gly Arg Glu Phe Trp Pro Gln Glu Val Trp Ser Arg Tyr Val 225 230 235 240 Lys Lys Leu Gly Asp Phe Ala Lys Pro Glu Asn Ile Asp Leu Ala Val 245 250 255 Gln Cys Leu Asn Glu Leu Ile Thr Asn Ala Leu His His Ile Pro Asp 260 265 270 Val Ile Thr Tyr Leu Ser Arg Leu Arg Asn Gln Ser Val Phe Asn Phe 275 280 285 Cys Ala Ile Pro Gln Val Met Ala Ile Ala Thr Leu Ala Ala Cys Tyr 290 295 300 Asn Asn Gln Gln Val Phe Lys Gly Ala Val Lys Ile Arg Lys Gly Gln 305 310 315 320 Ala Val Thr Leu Met Met Asp Ala Thr Asn Met Pro Ala Val Lys Ala 325 330 335 Ile Ile Tyr Gln Tyr Met Glu Glu Ile Tyr His Arg Ile Pro Asp Ser 340 345 350 Asp Pro Ser Ser Ser Lys Thr Arg Gln Ile Ile Ser Thr Ile Arg Thr 355 360 365 Gln Asn Leu Pro Asn Cys Gln Leu Ile Ser Arg Ser His Tyr Ser Pro 370 375 380 Ile Tyr Leu Ser Phe Val Met Leu Leu Ala Ala Leu Ser Trp Gln Tyr 385 390 395 400 Leu Thr Thr Leu Ser Gln Val Thr Glu Asp Tyr Val Gln Thr Gly Glu 405 410 415 His 6 417 PRT Human 6 Met Glu Phe Val Lys Cys Leu Gly His Pro Glu Glu Phe Tyr Asn Leu 1 5 10 15 Val Arg Phe Arg Ile Gly Gly Lys Arg Lys Val Met Pro Lys Met Asp 20 25 30 Gln Asp Ser Leu Ser Ser Ser Leu Lys Thr Cys Tyr Lys Tyr Leu Asn 35 40 45 Gln Thr Ser Arg Ser Phe Ala Ala Val Ile Gln Ala Leu Asp Gly Glu 50 55 60 Met Arg Asn Ala Val Cys Ile Phe Tyr Leu Val Leu Arg Ala Leu Asp 65 70 75 80 Thr Leu Glu Asp Asp Met Thr Ile Ser Val Glu Lys Lys Val Pro Leu 85 90 95 Leu His Asn Phe His Ser Phe Leu Tyr Gln Pro Asp Trp Arg Phe Met 100 105 110 Glu Ser Lys Glu Lys Asp Arg Gln Val Leu Glu Asp Phe Pro Thr Ile 115 120 125 Ser Leu Glu Phe Arg Asn Leu Ala Glu Lys Tyr Gln Thr Val Ile Ala 130 135 140 Asp Ile Cys Arg Arg Met Gly Ile Gly Met Ala Glu Phe Leu Asp Lys 145 150 155 160 His Val Thr Ser Glu Gln Glu Trp Asp Lys Tyr Cys His Tyr Val Ala 165 170 175 Gly Leu Val Gly Ile Gly Leu Ser Arg Leu Phe Ser Ala Ser Glu Phe 180 185 190 Glu Asp Pro Leu Val Gly Glu Asp Thr Glu Arg Ala Asn Ser Met Gly 195 200 205 Leu Phe Leu Gln Lys Thr Asn Ile Ile Arg Asp Tyr Leu Glu Asp Gln 210 215 220 Gln Gly Gly Arg Glu Phe Trp Pro Gln Glu Val Trp Ser Arg Tyr Val 225 230 235 240 Lys Lys Leu Gly Asp Phe Ala Lys Pro Glu Asn Ile Asp Leu Ala Val 245 250 255 Gln Cys Leu Asn Glu Leu Ile Thr Asn Ala Leu His His Ile Pro Asp 260 265 270 Val Ile Thr Tyr Leu Ser Arg Leu Arg Asn Gln Ser Val Phe Asn Phe 275 280 285 Cys Ala Ile Pro Gln Val Met Ala Ile Ala Thr Leu Ala Ala Cys Tyr 290 295 300 Asn Asn Gln Gln Val Phe Lys Gly Ala Val Lys Ile Arg Lys Gly Gln 305 310 315 320 Ala Val Thr Leu Met Met Asp Ala Thr Asn Met Pro Ala Val Lys Ala 325 330 335 Ile Ile Tyr Gln Tyr Met Glu Glu Ile Tyr His Arg Ile Pro Asp Ser 340 345 350 Asp Pro Ser Ser Ser Lys Thr Arg Gln Ile Ile Ser Thr Ile Arg Thr 355 360 365 Gln Asn Leu Pro Asn Cys Gln Leu Ile Ser Arg Ser His Tyr Ser Pro 370 375 380 Ile Tyr Leu Ser Phe Val Met Leu Leu Ala Ala Leu Ser Trp Gln Tyr 385 390 395 400 Leu Ala Thr Leu Ser Gln Val Thr Glu Asp Tyr Val Gln Thr Gly Glu 405 410 415 His 

That which is claimed is:
 1. An isolated peptide consisting of an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids.
 2. An isolated peptide comprising an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids.
 3. An isolated antibody that selectively binds to a peptide of claim
 2. 4. A method for producing any of the peptides of claim 1 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.
 5. A method for producing any of the peptides of claim 2 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.
 6. A method for detecting the presence of any of the peptides of claim 2 in a sample, said method comprising contacting said sample with a detection agent that specifically allows detection of the presence of the peptide in the sample and then detecting the presence of the peptide.
 7. A method for identifying a modulator of a peptide of claim 2, said method comprising contacting said peptide with an agent and determining if said agent has modulated the function or activity of said peptide.
 8. The method of claim 7, wherein said agent is administered to a host cell comprising an expression vector that expresses said peptide.
 9. A method for identifying an agent that binds to any of the peptides of claim 2, said method comprising contacting the peptide with an agent and assaying the contacted mixture to determine whether a complex is formed with the agent bound to the peptide.
 10. A pharmaceutical composition comprising an agent identified by the method of claim 9 and a pharmaceutically acceptable carrier therefor.
 11. A method for treating a disease or condition mediated by a human enzyme protein, said method comprising administering to a patient a pharmaceutically effective amount of an agent identified by the method of claim
 9. 12. A method for identifying a modulator of the expression of a peptide of claim 2, said method comprising contacting a cell expressing said peptide with an agent, and determining if said agent has modulated the expression of said peptide.
 13. An isolated human enzyme peptide having an amino acid sequence that shares at least 70% homology with an amino acid sequence shown in SEQ ID NO:2.
 14. A peptide according to claim 13 that shares at least 90 percent homology with an amino acid sequence shown in SEQ ID NO:2. 